Variant nucleic acid libraries for crth2

ABSTRACT

Provided herein are methods and compositions relating to prostaglandin D2 receptor 2 (DP2 or CRTH2R) libraries having nucleic acids encoding for a scaffold comprising a CRTH2R binding domain. CRTH2R libraries described herein encode for immunoglobulins including antibodies and single domain antibodies. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/904,595 filed on Sep. 23, 2019; U.S. ProvisionalPatent Application No. 62/935,590 filed on Nov. 14, 2019; and U.S.Provisional Patent Application No. 62/945,752 filed on Dec. 9, 2019,each of which is incorporated by reference in its entirety.

BACKGROUND

G protein-coupled receptors (GPCRs) such as Prostaglandin D2 receptor 2(DP2 or CRTH2R) are implicated in a wide variety of diseases. Raisingantibodies to GPCRs has been difficult due to problems in obtainingsuitable antigen because GPCRs are often expressed at low levels incells and are very unstable when purified. Thus, there is a need forimproved agents for therapeutic intervention which target GPCRs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are nucleic acid libraries, comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a CRTH2R binding immunoglobulin,wherein the CRTH2R binding immunoglobulin comprises a variant of aCRTH2R binding domain, wherein the CRTH2R binding domain is a ligand forthe CRTH2R, and wherein the nucleic acid library comprises at least10,000 variant immunoglobulin heavy chains and at least 10,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 50,000variant immunoglobulin heavy chains and at least 50,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 100,000variant immunoglobulin heavy chains and at least 100,000 variantimmunoglobulin light chains. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library comprises at least 10⁵non-identical nucleic acids. Further provided herein are nucleic acidlibraries, wherein a length of the immunoglobulin heavy chain whentranslated is about 90 to about 100 amino acids. Further provided hereinare nucleic acid libraries, wherein a length of the immunoglobulin heavychain when translated is about 100 to about 400 amino acids. Furtherprovided herein are nucleic acid libraries, wherein the variantimmunoglobulin heavy chain when translated comprises at least 80%sequence identity to any one of SEQ ID NO: 2338-2360 or 2403-2405.Further provided herein are nucleic acid libraries, wherein the variantimmunoglobulin light chain when translated comprises at least 80%sequence identity to any one of SEQ ID NO: 2361-2381 or 2406-2408.

Provided herein are nucleic acid libraries comprising a plurality ofnucleic acids, wherein each nucleic acid of the plurality of nucleicacids encodes for a sequence that when translated encodes for anantibody or antibody fragment thereof, wherein the antibody or antibodyfragment thereof comprises a variable region of a heavy chain (VH) thatcomprises a CRTH2R binding domain, wherein each nucleic acid of theplurality of nucleic acids comprises a sequence encoding for a sequencevariant of the CRTH2R binding domain, and wherein the antibody orantibody fragment binds to its antigen with a K_(D) of less than 100 nM.Further provided herein are nucleic acid libraries, wherein a length ofthe VH is about 90 to about 100 amino acids. Further provided herein arenucleic acid libraries, wherein a length of the VH is about 100 to about400 amino acids. Further provided herein are nucleic acid libraries,wherein a length of the VH is about 270 to about 300 base pairs. Furtherprovided herein are nucleic acid libraries, wherein a length of the VHis about 300 to about 1200 base pairs. Further provided herein arenucleic acid libraries, wherein the library comprises at least 10⁵non-identical nucleic acids.

Provided herein are nucleic acid libraries comprising: a plurality ofnucleic acids, wherein each of the nucleic acids encodes for a sequencethat when translated encodes for a CRTH2R single domain antibody,wherein each sequence of the plurality of sequences comprises a variantsequence encoding for a CDR1, CDR2, or CDR3 on a variable region of aheavy chain (VH); wherein the library comprises at least 30,000 variantsequences; and wherein the CRTH2R single domain antibody binds to itsantigen with a K_(D) of less than 100 nM. Further provided herein arenucleic acid libraries, wherein a length of the VH when translated isabout 90 to about 100 amino acids. Further provided herein are nucleicacid libraries, wherein a length of the VH when translated is about 100to about 400 amino acids. Further provided herein are nucleic acidlibraries, wherein a length of the VH is about 270 to about 300 basepairs. Further provided herein are nucleic acid libraries, wherein alength of the VH is about 300 to about 1200 base pairs. Further providedherein are nucleic acid libraries, wherein the VH when translatedcomprises at least 80% sequence identity to any one of SEQ ID NO:2338-2360 or 2403-2405.

Provided herein are antibodies or antibody fragments that bind CRTH2R,comprising an immunoglobulin heavy chain and an immunoglobulin lightchain: a. wherein the immunoglobulin heavy chain comprises an amino acidsequence at least about 90% identical to that set forth in any one ofSEQ ID NO: 2338-2360 or 2403-2405; and b. wherein the immunoglobulinlight chain comprises an amino acid sequence at least about 90%identical to that set forth in any one of SEQ ID NO: 2361-2381 or2406-2408. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2338; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2361. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2339; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2362. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2340; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2363. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2341; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2364. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2342; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2365. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2343; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2366. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2344; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2367. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2345; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2368. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2346; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2369. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2347; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2370. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2348; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2371. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2349; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2372. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2350; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2373. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2351; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2374. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2352; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2375. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2353; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2376. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2354; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2377. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2355; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2378. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2356; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2379. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2357; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2380. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2358; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2381. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2403; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2406. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2404; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2407. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2405; and wherein the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 2408. Further provided are antibodies or antibody fragments thatbind CRTH2R, wherein the antibody is a monoclonal antibody, a polyclonalantibody, a bi-specific antibody, a multispecific antibody, a graftedantibody, a human antibody, a humanized antibody, a synthetic antibody,a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), asingle chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fdfragment, a Fv fragment, a single-domain antibody, an isolatedcomplementarity determining region (CDR), a diabody, a fragmentcomprised of only a single monomeric variable domain, disulfide-linkedFvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or abantigen-binding fragments thereof. Further provided are antibodies orantibody fragments that bind CRTH2R, wherein the antibody or antibodyfragment thereof is chimeric or humanized. Further provided areantibodies or antibody fragments that bind CRTH2R, wherein the antibodyhas an EC50 less than about 25 nanomolar in a cAMP assay. Furtherprovided are antibodies or antibody fragments that bind CRTH2R, whereinthe antibody has an EC50 less than about 20 nanomolar in a cAMP assay.Further provided are antibodies or antibody fragments that bind CRTH2R,wherein the antibody has an EC50 less than about 10 nanomolar in a cAMPassay.

Provided herein are antibodies or antibody fragments, wherein theantibody or antibody fragment comprises a complementarity determiningregion (CDR) comprising an amino acid sequence at least about 90%identical to that set forth in any one of SEQ ID NOs: 2382-2402.

Provided herein are antibodies or antibody fragments, wherein theantibody or antibody fragment comprises a sequence of any one of SEQ IDNOs: 2382-2402 and wherein the antibody is a monoclonal antibody, apolyclonal antibody, a bi-specific antibody, a multispecific antibody, agrafted antibody, a human antibody, a humanized antibody, a syntheticantibody, a chimeric antibody, a camelized antibody, a single-chain Fvs(scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, aFd fragment, a Fv fragment, a single-domain antibody, an isolatedcomplementarity determining region (CDR), a diabody, a fragmentcomprised of only a single monomeric variable domain, disulfide-linkedFvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or abantigen-binding fragments thereof.

Provided herein are methods of treating a disease or disorder of thecentral nervous system, kidney, intestine, lung, hair, skin, bone, orcartilage, comprising administering the antibody or antibody fragmentdescribed herein.

Provided herein are methods of treating a disease or disordercharacterized by an inflammatory response, comprising administering theantibody or antibody fragment described herein.

Provided herein are methods of treating an allergic reaction, comprisingadministering the antibody or antibody fragment described herein.Further provided herein are methods, wherein the allergic reaction ischronic idiopathic urticaria or allergic rhinitis.

Provided herein are methods of treating asthma, comprising administeringthe antibody or antibody fragment described herein.

Provided herein are methods of treating alopecia or baldness, comprisingadministering the antibody or antibody fragment described herein.

Provided herein are methods for generating a nucleic acid libraryencoding for a CRTH2R antibody or antibody fragment thereof comprising:(a) providing predetermined sequences encoding for: i. a first pluralityof polynucleotides, wherein each polynucleotide of the first pluralityof polynucleotides encodes for at least 1000 variant sequence encodingfor CDR1 on a heavy chain; ii. a second plurality of polynucleotides,wherein each polynucleotide of the second plurality of polynucleotidesencodes for at least 1000 variant sequence encoding for CDR2 on a heavychain; iii. a third plurality of polynucleotides, wherein eachpolynucleotide of the third plurality of polynucleotides encodes for atleast 1000 variant sequence encoding for CDR3 on a heavy chain; and (b)mixing the first plurality of polynucleotides, the second plurality ofpolynucleotides, and the third plurality of polynucleotides to form thenucleic acid library of variant nucleic acids encoding for the CRTH2Rantibody or antibody fragment thereof, and wherein at least about 70% ofthe variant nucleic acids encode for an antibody or antibody fragmentthat binds to its antigen with a K_(D) of less than 100 nM. Furtherprovided herein are methods for generating a nucleic acid library,wherein the CRTH2R antibody or antibody fragment thereof is a singledomain antibody. Further provided herein are methods for generating anucleic acid library, wherein the single domain antibody comprises oneheavy chain variable domain. Further provided herein are methods forgenerating a nucleic acid library, wherein the single domain antibody isa VHH antibody. Further provided herein are methods for generating anucleic acid library, wherein the nucleic acid library comprises atleast 50,000 variant sequences. Further provided herein are methods forgenerating a nucleic acid library, wherein the nucleic acid librarycomprises at least 100,000 variant sequences. Further provided hereinare methods for generating a nucleic acid library, wherein the nucleicacid library comprises at least 10⁵ non-identical nucleic acids. Furtherprovided herein are methods for generating a nucleic acid library,wherein the nucleic acid library comprises at least one sequenceencoding for the CRTH2R antibody or antibody fragment that binds toCRTH2R with a K_(D) of less than 75 nM. Further provided herein aremethods for generating a nucleic acid library, wherein the nucleic acidlibrary comprises at least one sequence encoding for the CRTH2R antibodyor antibody fragment that binds to CRTH2R with a K_(D) of less than 50nM. Further provided herein are methods for generating a nucleic acidlibrary, wherein the nucleic acid library comprises at least onesequence encoding for the CRTH2R antibody or antibody fragment thatbinds to CRTH2R with a K_(D) of less than 10 nM. Further provided hereinare methods for generating a nucleic acid library, wherein the nucleicacid library comprises at least 500 variant sequences. Further providedherein are methods for generating a nucleic acid library, wherein thenucleic acid library comprises at least five sequences encoding for theCRTH2R antibody or antibody fragment that binds to CRTH2R with a K_(D)of less than 75 nM.

Provided herein are protein libraries encoded by the nucleic acidlibrary described herein, wherein the protein library comprisespeptides. Further provided herein are protein libraries, wherein theprotein library comprises immunoglobulins. Further provided herein areprotein libraries, wherein the protein library comprises antibodies.Further provided herein are protein libraries, wherein the proteinlibrary is a peptidomimetic library.

Provided herein are vector libraries comprising the nucleic acid librarydescribed herein.

Provided herein are cell libraries comprising the nucleic acid librarydescribed herein.

Provided herein are cell libraries comprising the protein librarydescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of G protein-coupled receptor (GPCR) ligandinteraction surfaces.

FIG. 2A depicts a first schematic of an immunoglobulin scaffold.

FIG. 2B depicts a second schematic of an immunoglobulin scaffold.

FIG. 3 depicts a schematic of a motif for placement in a scaffold.

FIG. 4 depicts a schematic of a GPCR.

FIG. 5 depicts schematics of segments for assembly of clonal fragmentsand non-clonal fragments.

FIG. 6 depicts schematics of segments for assembly of clonal fragmentsand non-clonal fragments.

FIG. 7 presents a diagram of steps demonstrating an exemplary processworkflow for gene synthesis as disclosed herein.

FIG. 8 illustrates an example of a computer system.

FIG. 9 is a block diagram illustrating an architecture of a computersystem.

FIG. 10 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 11 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIG. 12A depicts a schematic of an immunoglobulin scaffold comprising aVH domain attached to a VL domain using a linker.

FIG. 12B depicts a schematic of a full-domain architecture of animmunoglobulin scaffold comprising a VH domain attached to a VL domainusing a linker, a leader sequence, and pIII sequence.

FIG. 12C depicts a schematic of four framework elements (FW1, FW2, FW3,FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

FIG. 13 depicts a graph of yield of purified GPCR immunoglobulins.

FIG. 14 depicts FACS data of GPCR binding protein expression.

FIGS. 15A-15C depict cell-binding affinity of purified immunoglobulins.

FIG. 15D depicts cAMP activity of purified immunoglobulins.

FIG. 16 depicts BVP ELISA data of purified immunoglobulins.

FIGS. 17A-17B depict graphs of FACS analysis (FIG. 17A) and graphs of adose curve and cAMP activity (FIG. 17B) of CRTH2-41-51.

FIG. 18 depicts a graph of a dose curve of CRTH2-44-59.

FIG. 19 depicts a graph FACS analysis of CRTH2-44-59.

FIGS. 20A-20E depict FACS analysis plots of cell binding as measured bymean fluorescence intensity (MFI) vs. 8-point titrations with CRTH2R IgGusing CRTH2-74, CRTH2-24, CRTH2-28, CRTH2-39, CRTH2-19, CRTH2-9,CRTH2-8, CRTH2-27, CRTH2-45, CRTH2-35, CRTH2-50, CRTH2-66, CRTH2-57,CRTH2-32, CRTH2-15, CRTH2-25, CRTH2-42, CRTH2-55, CRTH2-60, andCRTH2-70.

FIG. 21A depicts an example gated dot plot showing CRTH2-27 binding at100 nM.

FIG. 21B depicts an example APC histogram showing CRTH2-27 binding at100 nM.

FIG. 22A depicts binding analysis as in previous figures usingcomparator antibody gPCR-51.

FIG. 22B depicts binding analysis as in previous figures usingcomparator antibody gPCR-52.

FIGS. 23A-23B depict IgG binding curves with CRTH2-9, CRTH2-27,CRTH2-50, CRTH2-32, and CRTH2-42, which have functional effects in cAMPassays.

FIG. 24A depicts results of CRTH2R cAMP assays across all antibodiestested at 300, 100, and 33 nM.

FIG. 24B depicts results of CRTH2R cAMP assays across all antibodiestested at 33 nM.

FIG. 25 indicates the negative allosteric effect seen in five of theCRTH2R IgG (CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, and CRTH2-42).

FIGS. 26A-26C depict control experiments of allosteric modulators,showing comparator antibody 52 is a positive allosteric modulator.

FIGS. 27A-27D depict activity of CRTH2R in β-arrestin recruitmentassays.

FIG. 28 depicts a schema of design of phage-displayed hyperimmunelibraries generated herein.

FIGS. 29A-29F depict graphs of binding affinity for the CRTH2Rimmunoglobulins CRTH2-48-03 (FIG. 29A), CRTH2-48-21 (FIG. 29B), andCRTH2-48-27 (FIG. 29C) and cAMP assays for CRTH2-48-03 (FIG. 29D),CRTH2-48-21 (FIG. 29E), and CRTH2-48-27 (FIG. 29F).

FIG. 30A depicts a schema of heavy chain IGHV3-23 design.

FIG. 30B depicts a schema of heavy chain IGHV1-69 design.

FIG. 30C depicts a schema of light chains IGKV 2-28 and IGLV 1-51design.

FIG. 30D depicts a schema of the theoretical diversity and finaldiversity of a GPCR library.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventionalmolecular biology techniques, which are within the skill of the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Definitions

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers+/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

Unless specifically stated, as used herein, the term “nucleic acid”encompasses double- or triple-stranded nucleic acids, as well assingle-stranded molecules. In double- or triple-stranded nucleic acids,the nucleic acid strands need not be coextensive (i.e., adouble-stranded nucleic acid need not be double-stranded along theentire length of both strands). Nucleic acid sequences, when provided,are listed in the 5′ to 3′ direction, unless stated otherwise. Methodsdescribed herein provide for the generation of isolated nucleic acids.Methods described herein additionally provide for the generation ofisolated and purified nucleic acids. A “nucleic acid” as referred toherein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, or more bases in length. Moreover, providedherein are methods for the synthesis of any number ofpolypeptide-segments encoding nucleotide sequences, including sequencesencoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomalpeptide-synthetase (NRPS) modules and synthetic variants, polypeptidesegments of other modular proteins, such as antibodies, polypeptidesegments from other protein families, including non-coding DNA or RNA,such as regulatory sequences e.g. promoters, transcription factors,enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived frommicroRNA, or any functional or structural DNA or RNA unit of interest.The following are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, intergenic DNA, loci(locus) defined from linkage analysis, exons, introns, messenger RNA(mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA,ribozymes, complementary DNA (cDNA), which is a DNA representation ofmRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or by amplification; DNA molecules produced synthetically or byamplification, genomic DNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. cDNAencoding for a gene or gene fragment referred herein may comprise atleast one region encoding for exon sequences without an interveningintron sequence in the genomic equivalent sequence.

As used herein, the term “percent (%) sequence identity” with respect toa sequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSSNEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

CRTH2R Libraries

Provided herein are methods and compositions relating to Gprotein-coupled receptor (GPCR) binding libraries for Prostaglandin D2receptor 2 (DP2 or CRTH2R) comprising nucleic acids encoding for ascaffold comprising a CRTH2R binding domain. Scaffolds as describedherein can stably support a CRTH2R binding domain. The CRTH2R bindingdomain may be designed based on surface interactions of a CRTH2R ligandand CRTH2R. Libraries as described herein may be further variegated toprovide for variant libraries comprising nucleic acids each encoding fora predetermined variant of at least one predetermined reference nucleicacid sequence. Further described herein are protein libraries that maybe generated when the nucleic acid libraries are translated. In someinstances, nucleic acid libraries as described herein are transferredinto cells to generate a cell library. Also provided herein aredownstream applications for the libraries synthesized using methodsdescribed herein. Downstream applications include identification ofvariant nucleic acids or protein sequences with enhanced biologicallyrelevant functions, e.g., improved stability, affinity, binding,functional activity, and for the treatment or prevention of a diseasestate associated with CRTH2R signaling.

Methods, compositions, and systems described herein for the optimizationof CRTH2R immunoglobulins or antibodies comprise a ratio-variantapproach that mirror the natural diversity of antibody sequences. Insome instances, libraries of optimized CRTH2R immunoglobulins orantibodies comprise variant CRTH2R immunoglobulin or antibody sequences.In some instances, the variant CRTH2R immunoglobulin or antibodysequences are designed comprising variant CDR regions. In someinstances, the variant CRTH2R immunoglobulin or antibody sequencescomprising variant CDR regions are generated by shuffling the naturalCDR sequences in a llama, humanized, or chimeric framework. In someinstances, such libraries are synthesized, cloned into expressionvectors, and translation products (antibodies) evaluated for activity.In some instances, fragments of sequences are synthesized andsubsequently assembled. In some instances, expression vectors are usedto display and enrich desired antibodies, such as phage display. In someinstances, the phage vector is a Fab phagemid vector. Selectionpressures used during enrichment in some instances includes bindingaffinity, toxicity, immunological tolerance, stability, or other factor.Such expression vectors allow antibodies with specific properties to beselected (“panning”), and subsequent propagation or amplification ofsuch sequences enriches the library with these sequences. Panning roundscan be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, ormore than 7 rounds. In some instances, each round of panning involves anumber of washes. In some instances, each round of panning involves atleast or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, ormore than 16 washes.

Described herein are methods and systems of in-silico library design.Libraries as described herein, in some instances, are designed based ona database comprising a variety of antibody sequences. In someinstances, the database comprises a plurality of variant antibodysequences against various targets. In some instances, the databasecomprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, or more than 5000 antibody sequences. An exemplary databaseis an iCAN database. In some instances, the database comprises naïve andmemory B-cell receptor sequences. In some instances, the naïve andmemory B-cell receptor sequences are human, mouse, or primate sequences.In some instances, the naïve and memory B-cell receptor sequences arehuman sequences. In some instances, the database is analyzed forposition specific variation. In some instances, antibodies describedherein comprise position specific variations in CDR regions. In someinstances, the CDR regions comprise multiple sites for variation.

Scaffold Libraries

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein sequences for CRTH2R binding domains are placed in thescaffold. Scaffold described herein allow for improved stability for arange of CRTH2R binding domain encoding sequences when inserted into thescaffold, as compared to an unmodified scaffold. Exemplary scaffoldsinclude, but are not limited to, a protein, a peptide, animmunoglobulin, derivatives thereof, or combinations thereof. In someinstances, the scaffold is an immunoglobulin. Scaffolds as describedherein comprise improved functional activity, structural stability,expression, specificity, or a combination thereof. In some instances,scaffolds comprise long regions for supporting a CRTH2R binding domain.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin. In some instances,the immunoglobulin is an antibody. As used herein, the term antibodywill be understood to include proteins having the characteristictwo-armed, Y-shape of a typical antibody molecule as well as one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Exemplary antibodies include, but are not limited to, amonoclonal antibody, a polyclonal antibody, a bi-specific antibody, amultispecific antibody, a grafted antibody, a human antibody, ahumanized antibody, a synthetic antibody, a chimeric antibody, acamelized antibody, a single-chain Fvs (scFv) (including fragments inwhich the VL and VH are joined using recombinant methods by a syntheticor natural linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules,including single chain Fab and scFab), a single chain antibody, a Fabfragment (including monovalent fragments comprising the VL, VH, CL, andCH1 domains), a F(ab′)2 fragment (including bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion), a Fd fragment (including fragments comprising the VH and CH1fragment), a Fv fragment (including fragments comprising the VL and VHdomains of a single arm of an antibody), a single-domain antibody (dAbor sdAb) (including fragments comprising a VH domain), an isolatedcomplementarity determining region (CDR), a diabody (including fragmentscomprising bivalent dimers such as two VL and VH domains bound to eachother and recognizing two different antigens), a fragment comprised ofonly a single monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. In some instances, the libraries disclosed hereincomprise nucleic acids encoding for a scaffold, wherein the scaffold isa Fv antibody, including Fv antibodies comprised of the minimum antibodyfragment which contains a complete antigen-recognition andantigen-binding site. In some embodiments, the Fv antibody consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association, and the three hypervariable regions of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. In some embodiments, the six hypervariableregions confer antigen-binding specificity to the antibody. In someembodiments, a single variable domain (or half of an Fv comprising onlythree hypervariable regions specific for an antigen, including singledomain antibodies isolated from camelid animals comprising one heavychain variable domain or variable region of a heavy chain such as VHHantibodies or nobodies) has the ability to recognize and bind antigen.In some instances, the libraries disclosed herein comprise nucleic acidsencoding for a scaffold, wherein the scaffold is a single-chain Fv orscFv, including antibody fragments comprising a VH, a VL, or both a VHand VL domain, wherein both domains are present in a single polypeptidechain. In some embodiments, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains allowing the scFv toform the desired structure for antigen binding. In some instances, ascFv is linked to the Fc fragment or a VHH is linked to the Fc fragment(including minibodies). In some instances, the antibody comprisesimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, e.g., molecules that contain an antigenbinding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1and IgA 2) or subclass.

In some embodiments, libraries comprise immunoglobulins that are adaptedto the species of an intended therapeutic target. Generally, thesemethods include “mammalization” and comprises methods for transferringdonor antigen-binding information to a less immunogenic mammal antibodyacceptor to generate useful therapeutic treatments. In some instances,the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee,baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, andhuman. In some instances, provided herein are libraries and methods forfelinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodiesthat contain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. In some instances,these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canineantigen-binding information from a donor antibody to a less immunogeniccanine antibody acceptor to generate treatments useful as therapeuticsin dogs. In some instances, caninized forms of non-canine antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-canine antibodies. In some instances, caninizedantibodies are canine antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-canine species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the canine antibody are replaced by corresponding non-canineFR residues. In some instances, caninized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The caninized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a canineantibody.

“Felinization” can comprise a method for transferring non-felineantigen-binding information from a donor antibody to a less immunogenicfeline antibody acceptor to generate treatments useful as therapeuticsin cats. In some instances, felinized forms of non-feline antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-feline antibodies. In some instances, felinizedantibodies are feline antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-feline species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the feline antibody are replaced by corresponding non-felineFR residues. In some instances, felinized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The felinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a felinizeantibody.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is a non-immunoglobulin. In someinstances, the scaffold is a non-immunoglobulin binding domain. Forexample, the scaffold is an antibody mimetic. Exemplary antibodymimetics include, but are not limited to, anticalins, affilins, affibodymolecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins,fynomers, Kunitz domain-based proteins, monobodies, anticalins,knottins, armadillo repeat protein-based proteins, and bicyclicpeptides.

Libraries described herein comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin, comprise variationsin at least one region of the immunoglobulin. Exemplary regions of theantibody for variation include, but are not limited to, acomplementarity-determining region (CDR), a variable domain, or aconstant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. Insome instances, the CDR is a heavy domain including, but not limited to,CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domainincluding, but not limited to, CDRL1, CDRL2, and CDRL3. In someinstances, the variable domain is variable domain, light chain (VL) orvariable domain, heavy chain (VH). In some instances, the CDR1, CDR2, orCDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3 of avariable domain, light chain (VL) can be referred to as CDRL1, CDRL2, orCDRL3, respectively. CDR1, CDR2, or CDR3 of a variable domain, heavychain (VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively.In some instances, the VL domain comprises kappa or lambda chains. Insome instances, the constant domain is constant domain, light chain (CL)or constant domain, heavy chain (CH).

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for a scaffold, wherein each nucleic acid encodesfor a predetermined variant of at least one predetermined referencenucleic acid sequence. In some cases, the predetermined referencesequence is a nucleic acid sequence encoding for a protein, and thevariant library comprises sequences encoding for variation of at least asingle codon such that a plurality of different variants of a singleresidue in the subsequent protein encoded by the synthesized nucleicacid are generated by standard translation processes. In some instances,the scaffold library comprises varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VHdomain. In some instances, the variant library comprises sequencesencoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons offramework element 1 (FW1), framework element 2 (FW2), framework element3 (FW3), or framework element 4 (FW4). An exemplary number of codons forvariation include, but are not limited to, at least or about 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin forvariation is from heavy chain V-gene family, heavy chain D-gene family,heavy chain J-gene family, light chain V-gene family, or light chainJ-gene family. In some instances, the light chain V-gene familycomprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda(IGL). Exemplary genes include, but are not limited to, IGHV1-18,IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28,IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28,IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, andIGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23,IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the geneis IGHV1-69 and IGHV3-30. In some instances, the gene is IGHJ3, IGHJ6,IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the gene isIGHJ3, IGHJ6, IGHJ, or IGHJ4.

Provided herein are libraries comprising nucleic acids encoding forimmunoglobulin scaffolds, wherein the libraries are synthesized withvarious numbers of fragments. In some instances, the fragments comprisethe CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In someinstances, the fragments comprise framework element 1 (FW1), frameworkelement 2 (FW2), framework element 3 (FW3), or framework element 4(FW4). In some instances, the scaffold libraries are synthesized with atleast or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, ormore than 5 fragments. The length of each of the nucleic acid fragmentsor average length of the nucleic acids synthesized may be at least orabout 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 basepairs. In some instances, the length is about 50 to 600, 75 to 575, 100to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for immunoglobulin scaffoldsas described herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 amino acids to about 75 amino acids. In some instances,the immunoglobulin scaffolds comprise at least or about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than5000 amino acids.

A number of variant sequences for the at least one region of theimmunoglobulin for variation are de novo synthesized using methods asdescribed herein. In some instances, a number of variant sequences is denovo synthesized for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH,or combinations thereof. In some instances, a number of variantsequences is de novo synthesized for framework element 1 (FW1),framework element 2 (FW2), framework element 3 (FW3), or frameworkelement 4 (FW4). The number of variant sequences may be at least orabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, or more than 500 sequences. In some instances, thenumber of variant sequences is at least or about 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000sequences. In some instances, the number of variant sequences is about10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325sequences.

Variant sequences for the at least one region of the immunoglobulin, insome instances, vary in length or sequence. In some instances, the atleast one region that is de novo synthesized is for CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances,the at least one region that is de novo synthesized is for frameworkelement 1 (FW1), framework element 2 (FW2), framework element 3 (FW3),or framework element 4 (FW4). In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acidsas compared to wild-type. In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, or 50 additional nucleotides or amino acids as comparedto wild-type. In some instances, the variant sequence comprises at leastor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 less nucleotides or amino acids as compared to wild-type. In someinstances, the libraries comprise at least or about 10¹, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ variants.

Following synthesis of scaffold libraries, scaffold libraries may beused for screening and analysis. For example, scaffold libraries areassayed for library displayability and panning. In some instances,displayability is assayed using a selectable tag. Exemplary tagsinclude, but are not limited to, a radioactive label, a fluorescentlabel, an enzyme, a chemiluminescent tag, a colorimetric tag, anaffinity tag or other labels or tags that are known in the art. In someinstances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA),or FLAG. In some instances, scaffold libraries are assayed by sequencingusing various methods including, but not limited to, single-moleculereal-time (SMRT) sequencing, Polony sequencing, sequencing by ligation,reversible terminator sequencing, proton detection sequencing, ionsemiconductor sequencing, nanopore sequencing, electronic sequencing,pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g.,Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the scaffold libraries are assayed for functionalactivity, structural stability (e.g., thermal stable or pH stable),expression, specificity, or a combination thereof. In some instances,the scaffold libraries are assayed for scaffolds capable of folding. Insome instances, a region of the antibody is assayed for functionalactivity, structural stability, expression, specificity, folding, or acombination thereof. For example, a VH region or VL region is assayedfor functional activity, structural stability, expression, specificity,folding, or a combination thereof.

CRTH2R Libraries

Provided herein are CRTH2R binding libraries comprising nucleic acidsencoding for scaffolds comprising sequences for CRTH2R binding domains.In some instances, the scaffolds are immunoglobulins. In some instances,the scaffolds comprising sequences for CRTH2R binding domains aredetermined by interactions between the CRTH2R binding domains and theCRTH2R.

Provided herein are libraries comprising nucleic acids encodingscaffolds comprising CRTH2R binding domains, wherein the CRTH2R bindingdomains are designed based on surface interactions on CRTH2R. In someinstances, the CRTH2R binding domain comprises a sequence as defined bySEQ ID NO: 1. In some instances, the CRTH2R binding domains interactwith the amino- or carboxy-terminus of the CRTH2R. In some instances,the CRTH2R binding domains interact with at least one transmembranedomain including, but not limited to, transmembrane domain 1 (TM1),transmembrane domain 2 (TM2), transmembrane domain 3 (TM3),transmembrane domain 4 (TM4), transmembrane domain 5 (TM5),transmembrane domain 6 (TM6), and transmembrane domain 7 (TM7). In someinstances, the CRTH2R binding domains interact with an intracellularsurface of the CRTH2R. For example, the CRTH2R binding domains interactwith at least one intracellular loop including, but not limited to,intracellular loop 1 (ICL1), intracellular loop 2 (ICL2), andintracellular loop 3 (ICL3). In some instances, the CRTH2R bindingdomains interact with an extracellular surface of the CRTH2R Forexample, the CRTH2R binding domains interact with at least oneextracellular domain (ECD) or extracellular loop (ECL) of the CRTH2R.The extracellular loops include, but are not limited to, extracellularloop 1 (ECL1), extracellular loop 2 (ECL2), and extracellular loop 3(ECL3).

Described herein are CRTH2R binding domains, wherein the CRTH2R bindingdomains are designed based on surface interactions between a CRTH2Rligand and the CRTH2R. In some instances, the ligand is a peptide. Insome instances, the ligand is a CRTH2R agonist. In some instances, theligand is a CRTH2R antagonist. In some instances, the ligand is a CRTH2Rallosteric modulator. In some instances, the allosteric modulator is anegative allosteric modulator. In some instances, the allostericmodulator is a positive allosteric modulator.

Sequences of CRTH2R binding domains based on surface interactionsbetween a CRTH2R ligand and the CRTH2R are analyzed using variousmethods. For example, multispecies computational analysis is performed.In some instances, a structure analysis is performed. In some instances,a sequence analysis is performed. Sequence analysis can be performedusing a database known in the art. Non-limiting examples of databasesinclude, but are not limited to, NCBI BLAST(blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC Genome Browser(genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPS Guide toPHARMACOLOGY (guidetopharmacology.org/).

Described herein are CRTH2R binding domains designed based on sequenceanalysis among various organisms. For example, sequence analysis isperformed to identify homologous sequences in different organisms.Exemplary organisms include, but are not limited to, mouse, rat, equine,sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan,monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human.

Following identification of CRTH2R binding domains, libraries comprisingnucleic acids encoding for the CRTH2R binding domains may be generated.In some instances, libraries of CRTH2R binding domains comprisesequences of CRTH2R binding domains designed based on conformationalligand interactions, peptide ligand interactions, small molecule ligandinteractions, extracellular domains of CRTH2R, or antibodies that targetCRTH2R. In some instances, libraries of CRTH2R binding domains comprisesequences of CRTH2R binding domains designed based on peptide ligandinteractions. Libraries of CRTH2R binding domains may be translated togenerate protein libraries. In some instances, libraries of CRTH2Rbinding domains are translated to generate peptide libraries,immunoglobulin libraries, derivatives thereof, or combinations thereof.In some instances, libraries of CRTH2R binding domains are translated togenerate protein libraries that are further modified to generatepeptidomimetic libraries. In some instances, libraries of CRTH2R bindingdomains are translated to generate protein libraries that are used togenerate small molecules.

Methods described herein provide for synthesis of libraries of CRTH2Rbinding domains comprising nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the libraries ofCRTH2R binding domains comprise varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon in a CRTH2R binding domain. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons ina CRTH2R binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for the CRTH2R binding domains, wherein thelibraries comprise sequences encoding for variation of length of theCRTH2R binding domains. In some instances, the library comprisessequences encoding for variation of length of at least or about 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less ascompared to a predetermined reference sequence. In some instances, thelibrary comprises sequences encoding for variation of length of at leastor about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or morethan 300 codons more as compared to a predetermined reference sequence.

Following identification of CRTH2R binding domains, the CRTH2R bindingdomains may be placed in scaffolds as described herein. In someinstances, the scaffolds are immunoglobulins. In some instances, theCRTH2R binding domains are placed in the CDRH3 region. CRTH2R bindingdomains that may be placed in scaffolds can also be referred to as amotif. Scaffolds comprising CRTH2R binding domains may be designed basedon binding, specificity, stability, expression, folding, or downstreamactivity. In some instances, the scaffolds comprising CRTH2R bindingdomains enable contact with the CRTH2R. In some instances, the scaffoldscomprising CRTH2R binding domains enables high affinity binding with theCRTH2R. An exemplary amino acid sequence of CRTH2R binding domain isdescribed in Table 1A.

TABLE 1A CRTH2R binding domain amino acid sequences SEQ ID NO GPCRAmino Acid Sequence 1 CRTH2R MSANATLKPLCPILEQMSRLQSHSNTSIRYIDHAAVLLHGLASLLGLVENGVILFVVGCRMRQTVVTTWVLHLALSDLLASASLPFFTYFLAVGHSWELGTTFCKLHSSIFFLNMFASGFLLSAISLDRCLQVVRPVWAQNHRTVAAAHKVCLVLWALAVLNTVPYFVFRDTISRLDGRIMCYYNVLLLNPGPDRDATCNSRQAALAVSKFLLAFLVPLAIIASSHAAVSLRLQHRGRRRPGRFVRLVAAVVAAFALCWGPYHVFSLLEARAHANPGLRPLVWRGLPFVTSLAFFNSVANPVLYVLTCPDMLRKLRRSLRTVLESVLVDDSELGGAGSSRRRRTSSTARSASPLALCSRP EEPRGPARLLGWLLGSCAASPQTGPLNRALSSTSS

Provided herein are scaffolds or immunoglobulins comprising CRTH2Rbinding domains, wherein the sequences of the CRTH2R binding domainssupport interaction with CRTH2R. The sequence may be homologous oridentical to a sequence of a CRTH2R ligand. In some instances, theCRTH2R binding domain sequence comprises at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 1. In some instances, the CRTH2R binding domainsequence comprises at least or about 95% homology to SEQ ID NO: 1. Insome instances, the CRTH2R binding domain sequence comprises at least orabout 97% homology to SEQ ID NO: 1. In some instances, the CRTH2Rbinding domain sequence comprises at least or about 99% homology to SEQID NO: 1. In some instances, the CRTH2R binding domain sequencecomprises at least or about 100% homology to SEQ ID NO: 1. In someinstances, the CRTH2R binding domain sequence comprises at least aportion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of SEQ ID NO: 1.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises complementarity determining regions (CDRs) comprising asequence as set forth in Table 1B and Table 14B. In some embodiments,the CRTH2R antibody or immunoglobulin sequence comprises complementaritydetermining regions (CDRs) comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 2409-2420 or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about95% homology to any one of SEQ ID NOs: 2409-2420 or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about97% homology to any one of SEQ ID NOs: 2409-2420 or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about99% homology to any one of SEQ ID NOs: 2409-2420 or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about100% homology to any one of SEQ ID NOs: 2409-2420 or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NOs: 2409-2420 or 2382-2402.

TABLE 1B Construct SEQ Description Amino Acid Sequence ID NOIGHV1-69 CDR1 GGTFSSYA 2409 IGHV1-69 CDR2 IIPIFGTA 2410 IGHV1-69 CDR3CARNNNNNNNNNFDYW 2411 IGHV3-23 CDR1 GFTFSSYA 2412 IGHV3-23 CDR2 ISGSGGST2413 IGHV3-23 CDR3 CAKNNNNNNNNNFDYW 2414 IGKV1-39 CDR1 QSISSY 2415IGKV1-39 CDR2 AAS 2416 IGKV1-39 CDR3 CQQSYSTPNTF 2417 IGKV3-20 CDR1QSVSSSY 2418 IGKV3-20 CDR2 GAS 2419 IGKV3-20 CDR3 CQQYGSSPNTF 2420

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDR1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anyone of SEQ ID NOs: 2409, 2412, 2415, or 2418. In some instances, theCRTH2R antibody or immunoglobulin sequence comprises CDR1 comprising atleast or about 95% homology to any one of SEQ ID NO: 2409, 2412, 2415,or 2418. In some instances, the CRTH2R antibody or immunoglobulinsequence comprises CDR1 comprising at least or about 97% homology to anyone of SEQ ID NO: 2409, 2412, 2415, or 2418. In some instances, theCRTH2R antibody or immunoglobulin sequence comprises CDR1 comprising atleast or about 99% homology to any one of SEQ ID NO: 2409, 2412, 2415,or 2418. In some instances, the CRTH2R antibody or immunoglobulinsequence comprises CDR1 comprising 100% homology to any one of SEQ IDNO: 2409, 2412, 2415, or 2418. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDR1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NO: 2409, 2412, 2415, or 2418.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDR2 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anyone of SEQ ID NOs: 2410, 2413, 2416, or 2419. In some instances, theCRTH2R antibody or immunoglobulin sequence comprises CDR2 comprising atleast or about 95% homology to any one of SEQ ID NO: 2410, 2413, 2416,or 2419. In some instances, the CRTH2R antibody or immunoglobulinsequence comprises CDR2 comprising at least or about 97% homology to anyone of SEQ ID NO: 2410, 2413, 2416, or 2419. In some instances, theCRTH2R antibody or immunoglobulin sequence comprises CDR2 comprising atleast or about 99% homology to any one of SEQ ID NO: 2410, 2413, 2416,or 2419. In some instances, the CRTH2R antibody or immunoglobulinsequence comprises CDR2 comprising at 100% homology to any one of SEQ IDNO: 2410, 2413, 2416, or 2419. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDR2 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NO: 2410, 2413, 2416, or 2419.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDR3 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anyone of SEQ ID NOs: 2411, 2414, 2417, 2420, or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises CDR3comprising at least or about 95% homology to any one of SEQ ID NO: 2411,2414, 2417, 2420, or 2382-2402. In some instances, the CRTH2R antibodyor immunoglobulin sequence comprises CDR3 comprising at least or about97% homology to any one of SEQ ID NO: 2411, 2414, 2417, 2420, or2382-2402. In some instances, the CRTH2R antibody or immunoglobulinsequence comprises CDR3 comprising at least or about 99% homology to anyone of SEQ ID NO: 2411, 2414, 2417, 2420, or 2382-2402. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises CDR3comprising 100% homology to any one of SEQ ID NO: 2411, 2414, 2417,2420, or 2382-2402. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDR3 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NO: 2411, 2414, 2417, 2420, or2382-2402.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2409; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2410; and a CDRH3 comprising at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2411. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2409; a CDRH2 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2410;and a CDRH3 comprising at least or about 95%, 97%, 99%, or 100% homologyto SEQ ID NO: 2411. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRH1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 2409; a CDRH2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 2410; and a CDRH3 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2411.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2412; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2413; and a CDRH3 comprising at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2414. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2412; a CDRH2 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2413;and a CDRH3 comprising at least or about 95%, 97%, 99%, or 100% homologyto SEQ ID NO: 2414. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRH1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 2412; a CDRH2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 2413; and a CDRH3 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2414.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2415; a CDRL2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2416; and a CDRL3 comprising at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2417. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRL1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2415; a CDRL2 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2416;and a CDRL3 comprising at least or about 95%, 97%, 99%, or 100% homologyto SEQ ID NO: 2417. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRL1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 2415; a CDRL2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 2416; and a CDRL3 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2417.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2418; a CDRL2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2419; and a CDRL3 comprising at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2420. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRL1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2418; a CDRL2 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2419;and a CDRL3 comprising at least or about 95%, 97%, 99%, or 100% homologyto SEQ ID NO: 2420. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises CDRL1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 2418; a CDRL2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 2419; and a CDRL3 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2420.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2409; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2410; a CDRH3 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2411, a CDRL1 comprising at least or about 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2415; a CDRL2 comprising at least orabout 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 2416; and a CDRL3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO: 2417. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2409; a CDRH2 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 2410; a CDRH3 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2411; a CDRL1 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2415; aCDRL2 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2416; and a CDRL3 comprising at least or about 95%, 97%, 99%,or 100% homology to SEQ ID NO: 2417. In some instances, the CRTH2Rantibody or immunoglobulin sequence comprises CDRH1 comprising at leasta portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,or more than 16 amino acids of SEQ ID NO: 2409; a CDRH2 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2410; a CDRH3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2411; aCDRL1 comprising at least a portion having at least or about 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2415;a CDRL2 comprising at least a portion having at least or about 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO:2416; and a CDRL3 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 2417.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2409; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2410; a CDRH3 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2411, a CDRL1 comprising at least or about 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2418; a CDRL2 comprising at least orabout 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 2419; and a CDRL3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO: 2420. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2409; a CDRH2 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 2410; a CDRH3 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2411; a CDRL1 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2418; aCDRL2 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2419; and a CDRL3 comprising at least or about 95%, 97%, 99%,or 100% homology to SEQ ID NO: 2420. In some instances, the CRTH2Rantibody or immunoglobulin sequence comprises CDRH1 comprising at leasta portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,or more than 16 amino acids of SEQ ID NO: 2409; a CDRH2 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2410; a CDRH3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2411; aCDRL1 comprising at least a portion having at least or about 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2418;a CDRL2 comprising at least a portion having at least or about 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO:2419; and a CDRL3 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 2420.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2412; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2413; a CDRH3 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2414, a CDRL1 comprising at least or about 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2415; a CDRL2 comprising at least orabout 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 2416; and a CDRL3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO: 2417. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2412; a CDRH2 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 2413; a CDRH3 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2414; a CDRL1 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2415; aCDRL2 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2416; and a CDRL3 comprising at least or about 95%, 97%, 99%,or 100% homology to SEQ ID NO: 2417. In some instances, the CRTH2Rantibody or immunoglobulin sequence comprises CDRH1 comprising at leasta portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,or more than 16 amino acids of SEQ ID NO: 2412; a CDRH2 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2413; a CDRH3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2414; aCDRL1 comprising at least a portion having at least or about 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2415;a CDRL2 comprising at least a portion having at least or about 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO:2416; and a CDRL3 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 2417.

In some embodiments, the CRTH2R antibody or immunoglobulin sequencecomprises a CDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 2412; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 2413; a CDRH3 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 2414, a CDRL1 comprising at least or about 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2418; a CDRL2 comprising at least orabout 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 2419; and a CDRL3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO: 2420. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2412; a CDRH2 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 2413; a CDRH3 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 2414; a CDRL1 comprisingat least or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 2418; aCDRL2 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 2419; and a CDRL3 comprising at least or about 95%, 97%, 99%,or 100% homology to SEQ ID NO: 2420. In some instances, the CRTH2Rantibody or immunoglobulin sequence comprises CDRH1 comprising at leasta portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,or more than 16 amino acids of SEQ ID NO: 2412; a CDRH2 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 2413; a CDRH3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2414; aCDRL1 comprising at least a portion having at least or about 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO: 2418;a CDRL2 comprising at least a portion having at least or about 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQ ID NO:2419; and a CDRL3 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 2420.

Described herein, in some embodiments, are antibodies or immunoglobulinsthat bind to the CRTH2R. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ IDNO: 2338-2360 or 2403-2405. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 95% sequence identity to any one of SEQ IDNO: 2338-2360 or 2403-2405. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNO: 2338-2360 or 2403-2405. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 99% sequence identity to any one of SEQ IDNO: 2338-2360 or 2403-2405. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 100% sequence identity to any one of SEQ IDNO: 2338-2360 or 2403-2405. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least a portion having at least or about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, or more than 400 amino acids of any one of SEQ ID NO: 2338-2360 and2403-2405.

In some instances, the CRTH2R antibody or immunoglobulin sequencecomprises a light chain variable domain comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NO: 2361-2381 or 2406-2408. Insome instances, the CRTH2R antibody or immunoglobulin sequence comprisesa light chain variable domain comprising at least or about 95% sequenceidentity to any one of SEQ ID NO: 2361-2381 and 2406-2408. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least or about 97% sequenceidentity to any one of SEQ ID NO: 2361-2381 or 2406-2408. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least or about 99% sequenceidentity to any one of SEQ ID NO: 2361-2381 or 2406-2408. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NO: 2361-2381 or 2406-2408. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least a portion having atleast or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids ofany one of SEQ ID NO: 2361-2381 or 2406-2408.

Provided herein are CRTH2R binding libraries comprising nucleic acidsencoding for scaffolds or immunoglobulins comprising CRTH2R bindingdomains comprise variation in domain type, domain length, or residuevariation. In some instances, the domain is a region in the scaffoldcomprising the CRTH2R binding domains. For example, the region is theVH, CDRH3, or VL domain. In some instances, the domain is the CRTH2Rbinding domain.

Methods described herein provide for synthesis of a CRTH2R bindinglibrary of nucleic acids each encoding for a predetermined variant of atleast one predetermined reference nucleic acid sequence. In some cases,the predetermined reference sequence is a nucleic acid sequence encodingfor a protein, and the variant library comprises sequences encoding forvariation of at least a single codon such that a plurality of differentvariants of a single residue in the subsequent protein encoded by thesynthesized nucleic acid are generated by standard translationprocesses. In some instances, the CRTH2R binding library comprisesvaried nucleic acids collectively encoding variations at multiplepositions. In some instances, the variant library comprises sequencesencoding for variation of at least a single codon of a VH, CDRH3, or VLdomain. In some instances, the variant library comprises sequencesencoding for variation of at least a single codon in a CRTH2R bindingdomain. For example, at least one single codon of a CRTH2R bindingdomain as listed in Table 1A is varied. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons ofa VH, CDRH3, or VL domain. In some instances, the variant librarycomprises sequences encoding for variation of multiple codons in aCRTH2R binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of a CRTH2R bindinglibrary of nucleic acids each encoding for a predetermined variant of atleast one predetermined reference nucleic acid sequence, wherein theCRTH2R binding library comprises sequences encoding for variation oflength of a domain. In some instances, the domain is VH, CDRH3, or VLdomain. In some instances, the domain is the CRTH2R binding domain. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons less as compared to a predeterminedreference sequence. In some instances, the library comprises sequencesencoding for variation of length of at least or about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200, 225, 250, 275, 300, or more than 300 codons more ascompared to a predetermined reference sequence.

Provided herein are CRTH2R binding libraries comprising nucleic acidsencoding for scaffolds comprising CRTH2R binding domains, wherein theCRTH2R binding libraries are synthesized with various numbers offragments. In some instances, the fragments comprise the VH, CDRH3, orVL domain. In some instances, the CRTH2R binding libraries aresynthesized with at least or about 2 fragments, 3 fragments, 4fragments, 5 fragments, or more than 5 fragments. The length of each ofthe nucleic acid fragments or average length of the nucleic acidssynthesized may be at least or about 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, or more than 600 base pairs. In some instances, the length isabout 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 basepairs.

CRTH2R binding libraries comprising nucleic acids encoding for scaffoldscomprising CRTH2R binding domains as described herein comprise variouslengths of amino acids when translated. In some instances, the length ofeach of the amino acid fragments or average length of the amino acidsynthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, or more than 150 amino acids. In some instances, thelength of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110,65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, thelength of the amino acid is about 22 to about 75 amino acids.

CRTH2R binding libraries comprising de novo synthesized variantsequences encoding for scaffolds comprising CRTH2R binding domainscomprise a number of variant sequences. In some instances, a number ofvariant sequences is de novo synthesized for a CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, VH, or a combination thereof. In someinstances, a number of variant sequences is de novo synthesized forframework element 1 (FW1), framework element 2 (FW2), framework element3 (FW3), or framework element 4 (FW4). In some instances, a number ofvariant sequences is de novo synthesized for a CRTH2R binding domain.For example, the number of variant sequences is about 1 to about 10sequences for the VH domain, about 10⁸ sequences for the CRTH2R bindingdomain, and about 1 to about 44 sequences for the VK domain. See FIGS.2A-2B. The number of variant sequences may be at least or about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, or more than 500 sequences. In some instances, the number ofvariant sequences is about 10 to 300, 25 to 275, 50 to 250, 75 to 225,100 to 200, or 125 to 150 sequences.

CRTH2R binding libraries comprising de novo synthesized variantsequences encoding for scaffolds comprising CRTH2R binding domainscomprise improved diversity. For example, variants are generated byplacing CRTH2R binding domain variants in immunoglobulin scaffoldvariants comprising N-terminal CDRH3 variations and C-terminal CDRH3variations. In some instances, variants include affinity maturationvariants. Alternatively or in combination, variants include variants inother regions of the immunoglobulin including, but not limited to,CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3. In some instances, the number ofvariants of the CRTH2R binding libraries is least or about 10⁴, 10⁵,10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷,10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ non-identical sequencesnon-identical sequences. For example, a library comprising about 10variant sequences for a VH region, about 237 variant sequences for aCDRH3 region, and about 43 variant sequences for a VL and CDRL3 regioncomprises 10⁵ non-identical sequences (10×237×43).

Provided herein are libraries comprising nucleic acids encoding for aCRTH2R antibody comprising variation in at least one region of theantibody, wherein the region is the CDR region. In some instances, theCRTH2R antibody is a single domain antibody comprising one heavy chainvariable domain such as a VHH antibody. In some instances, the VHHantibody comprises variation in one or more CDR regions. In someinstances, libraries described herein comprise at least or about 1, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or morethan 3000 sequences of a CDR1, CDR2, or CDR3. In some instances,libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸,10¹⁹, 10²⁰, or more than 10²⁰ sequences of a CDR1, CDR2, or CDR3. Forexample, the libraries comprise at least 2000 sequences of a CDR1, atleast 1200 sequences for CDR2, and at least 1600 sequences for CDR3. Insome instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain(VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VL. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise atleast 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2of the VL, and at least 140 sequences of a CDR3 of the VL. In someinstances, the libraries comprise at least 2 sequences of a CDR1 of theVL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequencesof a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9,IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14,IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In someinstances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain(VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VH. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise atleast 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2of the VH, and at least 10⁸ sequences of a CDR3 of the VH. In someinstances, the 20 or IGHV4-59/61. In some instances, the VH is IGHV1-69,IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In someinstances, the VH is IGHV1-69 and IGHV3-30. In some instances, the VH isIGHV3-23.

Libraries as described herein, in some embodiments, comprise varyinglengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3. In someinstances, the length of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, or more than 90 amino acids in length. For example, the CDRH3comprises at least or about 12, 15, 16, 17, 20, 21, or 23 amino acids inlength. In some instances, the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, orCDRH3 comprises a range of about 1 to about 10, about 5 to about 15,about 10 to about 20, or about 15 to about 30 amino acids in length.

Libraries comprising nucleic acids encoding for antibodies havingvariant CDR sequences as described herein comprise various lengths ofamino acids when translated. In some instances, the length of each ofthe amino acid fragments or average length of the amino acid synthesizedmay be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or more than 150 amino acids. In some instances, the length of theamino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105,70 to 100, or 75 to 95 amino acids. In some instances, the length of theamino acid is about 22 amino acids to about 75 amino acids. In someinstances, the antibodies comprise at least or about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000amino acids.

Ratios of the lengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3may vary in libraries described herein. In some instances, a CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprising at least or about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acidsin length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more than 90% of the library. For example, a CDRH3 comprising about23 amino acids in length is present in the library at 40%, a CDRH3comprising about 21 amino acids in length is present in the library at30%, a CDRH3 comprising about 17 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%. In some instances, a CDRH3 comprisingabout 20 amino acids in length is present in the library at 40%, a CDRH3comprising about 16 amino acids in length is present in the library at30%, a CDRH3 comprising about 15 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprisevariant CDR sequences that are shuffled to generate a library with atheoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences.

Provided herein are CRTH2R binding libraries encoding for animmunoglobulin. In some instances, the CRTH2R immunoglobulin is anantibody. In some instances, the CRTH2R immunoglobulin is a VHHantibody. In some instances, the CRTH2R immunoglobulin comprises abinding affinity (e.g., K_(D)) to CRTH2R of less than 1 nM, less than1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25nM, or less than 30 nM. In some instances, the CRTH2R immunoglobulincomprises a K_(D) of less than 1 nM. In some instances, the CRTH2Rimmunoglobulin comprises a K_(D) of less than 1.2 nM. In some instances,the CRTH2R immunoglobulin comprises a K_(D) of less than 2 nM. In someinstances, the CRTH2R immunoglobulin comprises a K_(D) of less than 5nM. In some instances, the CRTH2R immunoglobulin comprises a K_(D) ofless than 10 nM. In some instances, the CRTH2R immunoglobulin comprisesa K_(D) of less than 13.5 nM. In some instances, the CRTH2Rimmunoglobulin comprises a K_(D) of less than 15 nM. In some instances,the CRTH2R immunoglobulin comprises a K_(D) of less than 20 nM. In someinstances, the CRTH2R immunoglobulin comprises a K_(D) of less than 25nM. In some instances, the CRTH2R immunoglobulin comprises a K_(D) ofless than 30 nM.

In some instances, the CRTH2R immunoglobulin is a CRTH2R agonist. Insome instances, the CRTH2R immunoglobulin is a CRTH2R antagonist. Insome instances, the CRTH2R immunoglobulin is a CRTH2R allostericmodulator. In some instances, the allosteric modulator is a negativeallosteric modulator. In some instances, the allosteric modulator is apositive allosteric modulator. In some instances, the CRTH2Rimmunoglobulin results in agonistic, antagonistic, or allosteric effectsat a concentration of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120nM, 140 nM, 160 nM, 180 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700nM, 800 nM, 900 nM, 1000 nM, or more than 1000 nM. In some instances,the CRTH2R immunoglobulin is a negative allosteric modulator. In someinstances, the CRTH2R immunoglobulin is a negative allosteric modulatorat a concentration of at least or about 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. In some instances,the CRTH2R immunoglobulin is a negative allosteric modulator at aconcentration in a range of about 0.001 to about 100, 0.01 to about 90,about 0.1 to about 80, 1 to about 50, about 10 to about 40 nM, or about1 to about 10 nM. In some instances, the CRTH2R immunoglobulin comprisesan EC50 or IC50 of at least or about 0.001, 0.0025, 0.005, 0.01, 0.025,0.05, 0.06, 0.07, 0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6nM. In some instances, the CRTH2R immunoglobulin comprises an EC50 orIC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100nM.

CRTH2R immunoglobulins as described herein may comprise improvedproperties. In some instances, the CRTH2R immunoglobulins are monomeric.In some instances, the CRTH2R immunoglobulins are not prone toaggregation. In some instances, at least or about 70%, 75%, 80%, 85%,90%, 95%, or 99% of the CRTH2R immunoglobulins are monomeric. In someinstances, the CRTH2R immunoglobulins are thermostable. In someinstances, the CRTH2R immunoglobulins result in reduced non-specificbinding.

Following synthesis of CRTH2R binding libraries comprising nucleic acidsencoding scaffolds comprising CRTH2R binding domains, libraries may beused for screening and analysis. For example, libraries are assayed forlibrary displayability and panning. In some instances, displayability isassayed using a selectable tag. Exemplary tags include, but are notlimited to, a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag, an affinity tag or otherlabels or tags that are known in the art. In some instances, the tag ishistidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. For exampleas seen in FIG. 3, the CRTH2R binding libraries comprises nucleic acidsencoding scaffolds comprising CRTH2R binding domains with multiple tagssuch as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances,libraries are assayed by sequencing using various methods including, butnot limited to, single-molecule real-time (SMRT) sequencing, Polonysequencing, sequencing by ligation, reversible terminator sequencing,proton detection sequencing, ion semiconductor sequencing, nanoporesequencing, electronic sequencing, pyrosequencing, Maxam-Gilbertsequencing, chain termination (e.g., Sanger) sequencing, +S sequencing,or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising CRTH2R binding domains, wherein the libraries haveimproved specificity, stability, expression, folding, or downstreamactivity. In some instances, libraries described herein are used forscreening and analysis.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising CRTH2R binding domains, wherein the nucleic acidlibraries are used for screening and analysis. In some instances,screening and analysis comprises in vitro, in vivo, or ex vivo assays.Cells for screening include primary cells taken from living subjects orcell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) oreukaryotes (e.g., animals and plants). Exemplary animal cells include,without limitation, those from a mouse, rabbit, primate, and insect. Insome instances, cells for screening include a cell line including, butnot limited to, Chinese Hamster Ovary (CHO) cell line, human embryonickidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In someinstances, nucleic acid libraries described herein may also be deliveredto a multicellular organism. Exemplary multicellular organisms include,without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries or protein libraries encoded thereof describedherein may be screened for various pharmacological or pharmacokineticproperties. In some instances, the libraries are screened using in vitroassays, in vivo assays, or ex vivo assays. For example, in vitropharmacological or pharmacokinetic properties that are screened include,but are not limited to, binding affinity, binding specificity, andbinding avidity. Exemplary in vivo pharmacological or pharmacokineticproperties of libraries described herein that are screened include, butare not limited to, therapeutic efficacy, activity, preclinical toxicityproperties, clinical efficacy properties, clinical toxicity properties,immunogenicity, potency, and clinical safety properties.

Pharmacological or pharmacokinetic properties that may be screenedinclude, but are not limited to, cell binding affinity and cellactivity. For example, cell binding affinity assays or cell activityassays are performed to determine agonistic, antagonistic, or allostericeffects of libraries described herein. In some instances, the cellactivity assay is a cAMP assay. In some instances, libraries asdescribed herein are compared to cell binding or cell activity ofligands of CRTH2R.

Libraries as described herein may be screened in cell-based assays or innon-cell-based assays. Examples of non-cell-based assays include, butare not limited to, using viral particles, using in vitro translationproteins, and using protealiposomes with CRTH2R.

Nucleic acid libraries as described herein may be screened bysequencing. In some instances, next generation sequence is used todetermine sequence enrichment of CRTH2R binding variants. In someinstances, V gene distribution, J gene distribution, V gene family, CDR3counts per length, or a combination thereof is determined. In someinstances, clonal frequency, clonal accumulation, lineage accumulation,or a combination thereof is determined. In some instances, number ofsequences, sequences with VH clones, clones, clones greater than 1,clonotypes, clonotypes greater than 1, lineages, simpsons, or acombination thereof is determined. In some instances, a percentage ofnon-identical CDR3s is determined. For example, the percentage ofnon-identical CDR3s is calculated as the number of non-identical CDR3sin a sample divided by the total number of sequences that had a CDR3 inthe sample.

Provided herein are nucleic acid libraries, wherein the nucleic acidlibraries may be expressed in a vector. Expression vectors for insertingnucleic acid libraries disclosed herein may comprise eukaryotic orprokaryotic expression vectors. Exemplary expression vectors include,without limitation, mammalian expression vectors:pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG,pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 Vector,pEF1a-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro,pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors:pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plantexpression vectors: pRI 101-AN DNA and pCambia2301; and yeast expressionvectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1N5-His A andpDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in avector to generate a construct comprising a scaffold comprisingsequences of CRTH2R binding domains. In some instances, a size of theconstruct varies. In some instances, the construct comprises at least orabout 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700,1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than10000 bases. In some instances, a the construct comprises a range ofabout 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000,1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000,2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000,4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000,7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising CRTH2R binding domains, wherein the nucleic acidlibraries are expressed in a cell. In some instances, the libraries aresynthesized to express a reporter gene. Exemplary reporter genesinclude, but are not limited to, acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein,citrine fluorescent protein, orange fluorescent protein, cherryfluorescent protein, turquoise fluorescent protein, blue fluorescentprotein, horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), luciferase, and derivativesthereof. Methods to determine modulation of a reporter gene are wellknown in the art, and include, but are not limited to, fluorometricmethods (e.g. fluorescence spectroscopy, Fluorescence Activated CellSorting (FACS), fluorescence microscopy), and antibiotic resistancedetermination.

Diseases and Disorders

Provided herein are CRTH2R binding libraries comprising nucleic acidsencoding for scaffolds comprising CRTH2R binding domains that may havetherapeutic effects. In some instances, the CRTH2R binding librariesresult in protein when translated that is used to treat a disease ordisorder. In some instances, the protein is an immunoglobulin. In someinstances, the protein is a peptidomimetic. Exemplary diseases include,but are not limited to, cancer, inflammatory diseases or disorders, ametabolic disease or disorder, a cardiovascular disease or disorder, arespiratory disease or disorder, pain, a digestive disease or disorder,a reproductive disease or disorder, an endocrine disease or disorder, ora neurological disease or disorder. In some instances, the cancer is asolid cancer or a hematologic cancer. In some instances, an inhibitor ofprostaglandin D2 receptor 2 (DP2 or CRTH2R) as described herein is usedfor treatment of a disease or disorder of the central nervous system,kidney, intestine, lung, hair, skin, bone, or cartilage. In someinstances, an inhibitor or antagonist of CRTH2R as described herein isused for treatment of a disease or disorder characterized by aninflammatory response. In some instances, an inhibitor or antagonist ofCRTH2R as described herein is used for treatment of an allergicreaction. In some instances, the allergic reaction is chronic idiopathicurticaria. In some instances, the allergic reaction is allergicrhinitis. In some instances, an inhibitor or antagonist of CRTH2R asdescribed herein is used for treatment of asthma. In some instances, aninhibitor or antagonist of CRTH2R as described herein is used fortreatment of alopecia or baldness. In some instances, the subject is amammal. In some instances, the subject is a mouse, rabbit, dog, orhuman. Subjects treated by methods described herein may be infants,adults, or children. Pharmaceutical compositions comprising antibodiesor antibody fragments as described herein may be administeredintravenously or subcutaneously.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a pluralityof nucleic acids, wherein each nucleic acid encodes for a variant codonsequence compared to a reference nucleic acid sequence. In someinstances, each nucleic acid of a first nucleic acid population containsa variant at a single variant site. In some instances, the first nucleicacid population contains a plurality of variants at a single variantsite such that the first nucleic acid population contains more than onevariant at the same variant site. The first nucleic acid population maycomprise nucleic acids collectively encoding multiple codon variants atthe same variant site. The first nucleic acid population may comprisenucleic acids collectively encoding up to 19 or more codons at the sameposition. The first nucleic acid population may comprise nucleic acidscollectively encoding up to 60 variant triplets at the same position, orthe first nucleic acid population may comprise nucleic acidscollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation. Table 3 provides a listing ofeach codon possible (and the representative amino acid) for a variantsite.

TABLE 2 List of codons and amino acids One Three letter letter AminoAcids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGCTGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAGPhenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine HHis CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine LLeu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AACAAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine TThr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGGTyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectivelyencoding up to 20 codon variations at multiple positions. In such cases,each nucleic acid in the population comprises variation for codons atmore than one position in the same nucleic acid. In some instances, eachnucleic acid in the population comprises variation for codons at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecodons in a single nucleic acid. In some instances, each variant longnucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more codons in a single long nucleic acid. In someinstances, the variant nucleic acid population comprises variation forcodons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in asingle nucleic acid. In some instances, the variant nucleic acidpopulation comprises variation for codons in at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleicacid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization,parallelization, and vertical integration of the end-to-end process frompolynucleotide synthesis to gene assembly within nanowells on silicon tocreate a revolutionary synthesis platform. Devices described hereinprovide, with the same footprint as a 96-well plate, a silicon synthesisplatform is capable of increasing throughput by a factor of up to 1,000or more compared to traditional synthesis methods, with production of upto approximately 1,000,000 or more polynucleotides, or 10,000 or moregenes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomicdata has become an important factor for studies that delve into thebiological roles of various genes in both normal biology and diseasepathogenesis. At the core of this research is the central dogma ofmolecular biology and the concept of “residue-by-residue transfer ofsequential information.” Genomic information encoded in the DNA istranscribed into a message that is then translated into the protein thatis the active product within a given biological pathway.

Another exciting area of study is on the discovery, development andmanufacturing of therapeutic molecules focused on a highly-specificcellular target. High diversity DNA sequence libraries are at the coreof development pipelines for targeted therapeutics. Gene mutants areused to express proteins in a design, build, and test proteinengineering cycle that ideally culminates in an optimized gene for highexpression of a protein with high affinity for its therapeutic target.As an example, consider the binding pocket of a receptor. The ability totest all sequence permutations of all residues within the binding pocketsimultaneously will allow for a thorough exploration, increasing chancesof success. Saturation mutagenesis, in which a researcher attempts togenerate all possible mutations at a specific site within the receptor,represents one approach to this development challenge. Though costly andtime and labor-intensive, it enables each variant to be introduced intoeach position. In contrast, combinatorial mutagenesis, where a fewselected positions or short stretch of DNA may be modified extensively,generates an incomplete repertoire of variants with biasedrepresentation.

To accelerate the drug development pipeline, a library with the desiredvariants available at the intended frequency in the right positionavailable for testing—in other words, a precision library, enablesreduced costs as well as turnaround time for screening. Provided hereinare methods for synthesizing nucleic acid synthetic variant librarieswhich provide for precise introduction of each intended variant at thedesired frequency. To the end user, this translates to the ability tonot only thoroughly sample sequence space but also be able to querythese hypotheses in an efficient manner, reducing cost and screeningtime. Genome-wide editing can elucidate important pathways, librarieswhere each variant and sequence permutation can be tested for optimalfunctionality, and thousands of genes can be used to reconstruct entirepathways and genomes to re-engineer biological systems for drugdiscovery.

In a first example, a drug itself can be optimized using methodsdescribed herein. For example, to improve a specified function of anantibody, a variant polynucleotide library encoding for a portion of theantibody is designed and synthesized. A variant nucleic acid library forthe antibody can then be generated by processes described herein (e.g.,PCR mutagenesis followed by insertion into a vector). The antibody isthen expressed in a production cell line and screened for enhancedactivity. Example screens include examining modulation in bindingaffinity to an antigen, stability, or effector function (e.g., ADCC,complement, or apoptosis). Exemplary regions to optimize the antibodyinclude, without limitation, the Fc region, Fab region, variable regionof the Fab region, constant region of the Fab region, variable domain ofthe heavy chain or light chain (V_(H) or V_(L)), and specificcomplementarity-determining regions (CDRs) of V_(H) or V_(L).

Nucleic acid libraries synthesized by methods described herein may beexpressed in various cells associated with a disease state. Cellsassociated with a disease state include cell lines, tissue samples,primary cells from a subject, cultured cells expanded from a subject, orcells in a model system. Exemplary model systems include, withoutlimitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction ortreatment of a disease state, a variant nucleic acid library describedherein is expressed in a cell associated with a disease state, or one inwhich a cell a disease state can be induced. In some instances, an agentis used to induce a disease state in cells. Exemplary tools for diseasestate induction include, without limitation, a Cre/Lox recombinationsystem, LPS inflammation induction, and streptozotocin to inducehypoglycemia. The cells associated with a disease state may be cellsfrom a model system or cultured cells, as well as cells from a subjecthaving a particular disease condition. Exemplary disease conditionsinclude a bacterial, fungal, viral, autoimmune, or proliferativedisorder (e.g., cancer). In some instances, the variant nucleic acidlibrary is expressed in the model system, cell line, or primary cellsderived from a subject, and screened for changes in at least onecellular activity. Exemplary cellular activities include, withoutlimitation, proliferation, cycle progression, cell death, adhesion,migration, reproduction, cell signaling, energy production, oxygenutilization, metabolic activity, and aging, response to free radicaldamage, or any combination thereof

Substrates

Devices used as a surface for polynucleotide synthesis may be in theform of substrates which include, without limitation, homogenous arraysurfaces, patterned array surfaces, channels, beads, gels, and the like.Provided herein are substrates comprising a plurality of clusters,wherein each cluster comprises a plurality of loci that support theattachment and synthesis of polynucleotides. In some instances,substrates comprise a homogenous array surface. For example, thehomogenous array surface is a homogenous plate. The term “locus” as usedherein refers to a discrete region on a structure which provides supportfor polynucleotides encoding for a single predetermined sequence toextend from the surface. In some instances, a locus is on a twodimensional surface, e.g., a substantially planar surface. In someinstances, a locus is on a three-dimensional surface, e.g., a well,microwell, channel, or post. In some instances, a surface of a locuscomprises a material that is actively functionalized to attach to atleast one nucleotide for polynucleotide synthesis, or preferably, apopulation of identical nucleotides for synthesis of a population ofpolynucleotides. In some instances, polynucleotide refers to apopulation of polynucleotides encoding for the same nucleic acidsequence. In some cases, a surface of a substrate is inclusive of one ora plurality of surfaces of a substrate. The average error rates forpolynucleotides synthesized within a library described here using thesystems and methods provided are often less than 1 in 1000, less thanabout 1 in 2000, less than about 1 in 3000 or less often without errorcorrection.

Provided herein are surfaces that support the parallel synthesis of aplurality of polynucleotides having different predetermined sequences ataddressable locations on a common support. In some instances, asubstrate provides support for the synthesis of more than 50, 100, 200,400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000;20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000;1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000;4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides.In some cases, the surfaces provide support for the synthesis of morethan 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000;5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000;500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000;1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000;3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or morepolynucleotides encoding for distinct sequences. In some instances, atleast a portion of the polynucleotides have an identical sequence or areconfigured to be synthesized with an identical sequence. In someinstances, the substrate provides a surface environment for the growthof polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinctloci of a substrate, wherein each locus supports the synthesis of apopulation of polynucleotides. In some cases, each locus supports thesynthesis of a population of polynucleotides having a different sequencethan a population of polynucleotides grown on another locus. In someinstances, each polynucleotide sequence is synthesized with 1, 2, 3, 4,5, 6, 7, 8, 9 or more redundancy across different loci within the samecluster of loci on a surface for polynucleotide synthesis. In someinstances, the loci of a substrate are located within a plurality ofclusters. In some instances, a substrate comprises at least 10, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.In some instances, a substrate comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some instances, a substratecomprises about 10,000 distinct loci. The amount of loci within a singlecluster is varied in different instances. In some cases, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances,each cluster includes about 50-500 loci. In some instances, each clusterincludes about 100-200 loci. In some instances, each cluster includesabout 100-150 loci. In some instances, each cluster includes about 109,121, 130 or 137 loci. In some instances, each cluster includes about 19,20, 61, 64 or more loci. Alternatively or in combination, polynucleotidesynthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized ona substrate is dependent on the number of distinct loci available in thesubstrate. In some instances, the density of loci within a cluster orsurface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100,130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In somecases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500,10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distancebetween the centers of two adjacent loci within a cluster or surface isfrom about 10-500, from about 10-200, or from about 10-100 um. In someinstances, the distance between two centers of adjacent loci is greaterthan about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In someinstances, the distance between the centers of two adjacent loci is lessthan about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, eachlocus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is atleast or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 clusterper 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50clusters per 1 mm² or more. In some instances, a substrate comprisesfrom about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In someinstances, the distance between the centers of two adjacent clusters isat least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10,0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, eachcluster has a cross section of about 0.5 to about 2, about 0.5 to about1, or about 1 to about 2 mm. In some cases, each cluster has a crosssection of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interiorcross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 wellplate, for example between about 100 and about 200 mm by between about50 and about 150 mm. In some instances, a substrate has a diameter lessthan or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or50 mm. In some instances, the diameter of a substrate is between about25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In someinstances, a substrate has a planar surface area of at least about 100;200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000;40,000; 50,000 mm² or more. In some instances, the thickness of asubstrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated fromany variety of materials suitable for the methods, compositions, andsystems described herein. In certain instances, substrate materials arefabricated to exhibit a low level of nucleotide binding. In someinstances, substrate materials are modified to generate distinctsurfaces that exhibit a high level of nucleotide binding. In someinstances, substrate materials are transparent to visible and/or UVlight. In some instances, substrate materials are sufficientlyconductive, e.g., are able to form uniform electric fields across all ora portion of a substrate. In some instances, conductive materials areconnected to an electric ground. In some instances, the substrate isheat conductive or insulated. In some instances, the materials arechemical resistant and heat resistant to support chemical or biochemicalreactions, for example polynucleotide synthesis reaction processes. Insome instances, a substrate comprises flexible materials. For flexiblematerials, materials can include, without limitation: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like. Insome instances, a substrate comprises rigid materials. For rigidmaterials, materials can include, without limitation: glass; fusesilica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). Thesubstrate, solid support or reactors can be fabricated from a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane(PDMS), and glass. The substrates/solid supports or the microstructures,reactors therein may be manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates have a surfacearchitecture suitable for the methods, compositions, and systemsdescribed herein. In some instances, a substrate comprises raised and/orlowered features. One benefit of having such features is an increase insurface area to support polynucleotide synthesis. In some instances, asubstrate having raised and/or lowered features is referred to as athree-dimensional substrate. In some cases, a three-dimensionalsubstrate comprises one or more channels. In some cases, one or moreloci comprise a channel. In some cases, the channels are accessible toreagent deposition via a deposition device such as a material depositiondevice. In some cases, reagents and/or fluids collect in a larger wellin fluid communication one or more channels. For example, a substratecomprises a plurality of channels corresponding to a plurality of lociwith a cluster, and the plurality of channels are in fluid communicationwith one well of the cluster. In some methods, a library ofpolynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates are configured forpolynucleotide synthesis. In some instances, the structure is configuredto allow for controlled flow and mass transfer paths for polynucleotidesynthesis on a surface. In some instances, the configuration of asubstrate allows for the controlled and even distribution of masstransfer paths, chemical exposure times, and/or wash efficacy duringpolynucleotide synthesis. In some instances, the configuration of asubstrate allows for increased sweep efficiency, for example byproviding sufficient volume for a growing polynucleotide such that theexcluded volume by the growing polynucleotide does not take up more than50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1%, or less of the initially available volume that is available orsuitable for growing the polynucleotide. In some instances, athree-dimensional structure allows for managed flow of fluid to allowfor the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates comprise structuressuitable for the methods, compositions, and systems described herein. Insome instances, segregation is achieved by physical structure. In someinstances, segregation is achieved by differential functionalization ofthe surface generating active and passive regions for polynucleotidesynthesis. In some instances, differential functionalization is achievedby alternating the hydrophobicity across the substrate surface, therebycreating water contact angle effects that cause beading or wetting ofthe deposited reagents. Employing larger structures can decreasesplashing and cross-contamination of distinct polynucleotide synthesislocations with reagents of the neighboring spots. In some cases, adevice, such as a material deposition device, is used to depositreagents to distinct polynucleotide synthesis locations. Substrateshaving three-dimensional features are configured in a manner that allowsfor the synthesis of a large number of polynucleotides (e.g., more thanabout 10,000) with a low error rate (e.g., less than about 1:500,1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases,a substrate comprises features with a density of about or greater thanabout 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height,and/or volume as another well of the substrate. A channel of a substratemay have the same or different width, height, and/or volume as anotherchannel of the substrate. In some instances, the diameter of a clusteror the diameter of a well comprising a cluster, or both, is betweenabout 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1,0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or0.5-2 mm. In some instances, the diameter of a cluster or well or bothis less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06,or 0.05 mm. In some instances, the diameter of a cluster or well or bothis between about 1.0 and 1.3 mm. In some instances, the diameter of acluster or well, or both is about 1.150 mm. In some instances, thediameter of a cluster or well, or both is about 0.08 mm. The diameter ofa cluster refers to clusters within a two-dimensional orthree-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000,100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases,the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channelscorresponding to a plurality of loci within a cluster, wherein theheight or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50,or 10-50 um. In some cases, the height of a channel is less than 100,80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in asubstantially planar substrate) or both channel and locus (e.g., in athree-dimensional substrate wherein a locus corresponds to a channel) isfrom about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, forexample, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the diameter of a channel, locus, or both channel and locusis less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the distance between the center of two adjacent channels,loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200,5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface,wherein the surface comprises various surface modifications. In someinstances, the surface modifications are employed for the chemicaland/or physical alteration of a surface by an additive or subtractiveprocess to change one or more chemical and/or physical properties of asubstrate surface or a selected site or region of a substrate surface.For example, surface modifications include, without limitation, (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

In some cases, the addition of a chemical layer on top of a surface(referred to as adhesion promoter) facilitates structured patterning ofloci on a surface of a substrate. Exemplary surfaces for application ofadhesion promotion include, without limitation, glass, silicon, silicondioxide and silicon nitride. In some cases, the adhesion promoter is achemical with a high surface energy. In some instances, a secondchemical layer is deposited on a surface of a substrate. In some cases,the second chemical layer has a low surface energy. In some cases,surface energy of a chemical layer coated on a surface supportslocalization of droplets on the surface. Depending on the patterningarrangement selected, the proximity of loci and/or area of fluid contactat the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto whichnucleic acids or other moieties are deposited, e.g., for polynucleotidesynthesis, are smooth or substantially planar (e.g., two-dimensional) orhave irregularities, such as raised or lowered features (e.g.,three-dimensional features). In some instances, a substrate surface ismodified with one or more different layers of compounds. Suchmodification layers of interest include, without limitation, inorganicand organic layers such as metals, metal oxides, polymers, small organicmolecules and the like.

In some instances, resolved loci of a substrate are functionalized withone or more moieties that increase and/or decrease surface energy. Insome cases, a moiety is chemically inert. In some cases, a moiety isconfigured to support a desired chemical reaction, for example, one ormore processes in a polynucleotide synthesis reaction. The surfaceenergy, or hydrophobicity, of a surface is a factor for determining theaffinity of a nucleotide to attach onto the surface. In some instances,a method for substrate functionalization comprises: (a) providing asubstrate having a surface that comprises silicon dioxide; and (b)silanizing the surface using, a suitable silanizing agent describedherein or otherwise known in the art, for example, an organofunctionalalkoxysilane molecule. Methods and functionalizing agents are describedin U.S. Pat. No. 5,474,796, which is herein incorporated by reference inits entirety.

In some instances, a substrate surface is functionalized by contact witha derivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the substratesurface, typically via reactive hydrophilic moieties present on thesubstrate surface. Silanization generally covers a surface throughself-assembly with organofunctional alkoxysilane molecules. A variety ofsiloxane functionalizing reagents can further be used as currently knownin the art, e.g., for lowering or increasing surface energy. Theorganofunctional alkoxysilanes are classified according to their organicfunctions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis mayinclude processes involving phosphoramidite chemistry. In someinstances, polynucleotide synthesis comprises coupling a base withphosphoramidite. Polynucleotide synthesis may comprise coupling a baseby deposition of phosphoramidite under coupling conditions, wherein thesame base is optionally deposited with phosphoramidite more than once,i.e., double coupling. Polynucleotide synthesis may comprise capping ofunreacted sites. In some instances, capping is optional. Polynucleotidesynthesis may also comprise oxidation or an oxidation step or oxidationsteps. Polynucleotide synthesis may comprise deblocking, detritylation,and sulfurization. In some instances, polynucleotide synthesis compriseseither oxidation or sulfurization. In some instances, between one oreach step during a polynucleotide synthesis reaction, the device iswashed, for example, using tetrazole or acetonitrile. Time frames forany one step in a phosphoramidite synthesis method may be less thanabout 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise asubsequent addition of a phosphoramidite building block (e.g.,nucleoside phosphoramidite) to a growing polynucleotide chain for theformation of a phosphite triester linkage. Phosphoramiditepolynucleotide synthesis proceeds in the 3′ to 5′ direction.Phosphoramidite polynucleotide synthesis allows for the controlledaddition of one nucleotide to a growing nucleic acid chain per synthesiscycle. In some instances, each synthesis cycle comprises a couplingstep. Phosphoramidite coupling involves the formation of a phosphitetriester linkage between an activated nucleoside phosphoramidite and anucleoside bound to the substrate, for example, via a linker. In someinstances, the nucleoside phosphoramidite is provided to the deviceactivated. In some instances, the nucleoside phosphoramidite is providedto the device with an activator. In some instances, nucleosidephosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition of a nucleosidephosphoramidite, the device is optionally washed. In some instances, thecoupling step is repeated one or more additional times, optionally witha wash step between nucleoside phosphoramidite additions to thesubstrate. In some instances, a polynucleotide synthesis method usedherein comprises 1, 2, 3 or more sequential coupling steps. Prior tocoupling, in many cases, the nucleoside bound to the device isde-protected by removal of a protecting group, where the protectinggroup functions to prevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step is usefulto block unreacted substrate-bound 5′-OH groups after coupling fromfurther chain elongation, preventing the formation of polynucleotideswith internal base deletions. Further, phosphoramidites activated with1H-tetrazole may react, to a small extent, with the O6 position ofguanosine. Without being bound by theory, upon oxidation with I₂/water,this side product, possibly via O6-N7 migration, may undergodepurination. The apurinic sites may end up being cleaved in the courseof the final deprotection of the polynucleotide thus reducing the yieldof the full-length product. The O6 modifications may be removed bytreatment with the capping reagent prior to oxidation with I₂/water. Insome instances, inclusion of a capping step during polynucleotidesynthesis decreases the error rate as compared to synthesis withoutcapping. As an example, the capping step comprises treating thesubstrate-bound polynucleotide with a mixture of acetic anhydride and1-methylimidazole. Following a capping step, the device is optionallywashed.

In some instances, following addition of a nucleoside phosphoramidite,and optionally after capping and one or more wash steps, the devicebound growing nucleic acid is oxidized. The oxidation step comprises thephosphite triester is oxidized into a tetracoordinated phosphatetriester, a protected precursor of the naturally occurring phosphatediester internucleoside linkage. In some instances, oxidation of thegrowing polynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base (e.g., pyridine, lutidine,collidine). Oxidation may be carried out under anhydrous conditionsusing, e.g. tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for device drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, the deviceand growing polynucleotide is optionally washed. In some instances, thestep of oxidation is substituted with a sulfurization step to obtainpolynucleotide phosphorothioates, wherein any capping steps can beperformed after the sulfurization. Many reagents are capable of theefficient sulfur transfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, the protected 5′ end of the device bound growingpolynucleotide is removed so that the primary hydroxyl group is reactivewith a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound polynucleotide and thus reduces theyield of the desired full-length product. Methods and compositions ofthe disclosure described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the device bound polynucleotide is washed after deblocking. In someinstances, efficient washing after deblocking contributes to synthesizedpolynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve aniterating sequence of the following steps: application of a protectedmonomer to an actively functionalized surface (e.g., locus) to link witheither the activated surface, a linker or with a previously deprotectedmonomer; deprotection of the applied monomer so that it is reactive witha subsequently applied protected monomer; and application of anotherprotected monomer for linking. One or more intermediate steps includeoxidation or sulfurization. In some instances, one or more wash stepsprecede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise aseries of chemical steps. In some instances, one or more steps of asynthesis method involve reagent cycling, where one or more steps of themethod comprise application to the device of a reagent useful for thestep. For example, reagents are cycled by a series of liquid depositionand vacuum drying steps. For substrates comprising three-dimensionalfeatures such as wells, microwells, channels and the like, reagents areoptionally passed through one or more regions of the device via thewells and/or channels.

Methods and systems described herein relate to polynucleotide synthesisdevices for the synthesis of polynucleotides. The synthesis may be inparallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or morepolynucleotides can be synthesized in parallel. The total numberpolynucleotides that may be synthesized in parallel may be from2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700,11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250,20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill inthe art appreciate that the total number of polynucleotides synthesizedin parallel may fall within any range bound by any of these values, forexample 25-100. The total number of polynucleotides synthesized inparallel may fall within any range defined by any of the values servingas endpoints of the range. Total molar mass of polynucleotidessynthesized within the device or the molar mass of each of thepolynucleotides may be at least or at least about 10, 20, 30, 40, 50,100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The lengthof each of the polynucleotides or average length of the polynucleotideswithin the device may be at least or about at least 10, 15, 20, 25, 30,35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. Thelength of each of the polynucleotides or average length of thepolynucleotides within the device may be at most or about at most 500,400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10 nucleotides, or less. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100,15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciatethat the length of each of the polynucleotides or average length of thepolynucleotides within the device may fall within any range bound by anyof these values, for example 100-300. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall within any range defined by any of the values serving asendpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allowfor synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175,200 nucleotides per hour, or more are synthesized. Nucleotides includeadenine, guanine, thymine, cytosine, uridine building blocks, oranalogs/modified versions thereof. In some instances, libraries ofpolynucleotides are synthesized in parallel on substrate. For example, adevice comprising about or at least about 100; 1,000; 10,000; 30,000;75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or5,000,000 resolved loci is able to support the synthesis of at least thesame number of distinct polynucleotides, wherein polynucleotide encodinga distinct sequence is synthesized on a resolved locus. In someinstances, a library of polynucleotides is synthesized on a device withlow error rates described herein in less than about three months, twomonths, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acidsassembled from a polynucleotide library synthesized with low error rateusing the substrates and methods described herein are prepared in lessthan about three months, two months, one month, three weeks, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of alibrary of nucleic acids comprising variant nucleic acids differing at aplurality of codon sites. In some instances, a nucleic acid may have 1site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may beadjacent. In some instances, the one or more sites of variant codonsites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variantcodon sites, wherein all the variant codon sites are adjacent to oneanother, forming a stretch of variant codon sites. In some instances, anucleic acid may comprise multiple sites of variant codon sites, whereinnone the variant codon sites are adjacent to one another. In someinstances, a nucleic acid may comprise multiple sites of variant codonsites, wherein some the variant codon sites are adjacent to one another,forming a stretch of variant codon sites, and some of the variant codonsites are not adjacent to one another.

Referring to the Figures, FIG. 7 illustrates an exemplary processworkflow for synthesis of nucleic acids (e.g., genes) from shorternucleic acids. The workflow is divided generally into phases: (1) denovo synthesis of a single stranded nucleic acid library, (2) joiningnucleic acids to form larger fragments, (3) error correction, (4)quality control, and (5) shipment. Prior to de novo synthesis, anintended nucleic acid sequence or group of nucleic acid sequences ispreselected. For example, a group of genes is preselected forgeneration.

Once large nucleic acids for generation are selected, a predeterminedlibrary of nucleic acids is designed for de novo synthesis. Varioussuitable methods are known for generating high density polynucleotidearrays. In the workflow example, a device surface layer is provided. Inthe example, chemistry of the surface is altered in order to improve thepolynucleotide synthesis process. Areas of low surface energy aregenerated to repel liquid while areas of high surface energy aregenerated to attract liquids. The surface itself may be in the form of aplanar surface or contain variations in shape, such as protrusions ormicrowells which increase surface area. In the workflow example, highsurface energy molecules selected serve a dual function of supportingDNA chemistry, as disclosed in International Patent ApplicationPublication WO/2015/021080, which is herein incorporated by reference inits entirety.

In situ preparation of polynucleotide arrays is generated on a solidsupport 701 and utilizes single nucleotide extension process to extendmultiple oligomers in parallel. A deposition device, such as a materialdeposition device, is designed to release reagents in a step wisefashion such that multiple polynucleotides extend, in parallel, oneresidue at a time to generate oligomers with a predetermined nucleicacid sequence 702. In some instances, polynucleotides are cleaved fromthe surface at this stage. Cleavage includes gas cleavage, e.g., withammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber.In this exemplary workflow, the reaction chamber (also referred to as“nanoreactor”) is a silicon coated well, containing PCR reagents andlowered onto the polynucleotide library 703. Prior to or after thesealing 704 of the polynucleotides, a reagent is added to release thepolynucleotides from the substrate. In the exemplary workflow, thepolynucleotides are released subsequent to sealing of the nanoreactor705. Once released, fragments of single stranded polynucleotideshybridize in order to span an entire long range sequence of DNA. Partialhybridization 705 is possible because each synthesized polynucleotide isdesigned to have a small portion overlapping with at least one otherpolynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerasecycles, the polynucleotides anneal to complementary fragments and gapsare filled in by a polymerase. Each cycle increases the length ofvarious fragments randomly depending on which polynucleotides find eachother. Complementarity amongst the fragments allows for forming acomplete large span of double stranded DNA 706.

After PCA is complete, the nanoreactor is separated from the device 707and positioned for interaction with a device having primers for PCR 708.After sealing, the nanoreactor is subject to PCR 709 and the largernucleic acids are amplified. After PCR 710, the nanochamber is opened711, error correction reagents are added 712, the chamber is sealed 713and an error correction reaction occurs to remove mismatched base pairsand/or strands with poor complementarity from the double stranded PCRamplification products 714. The nanoreactor is opened and separated 715.Error corrected product is next subject to additional processing steps,such as PCR and molecular bar coding, and then packaged 722 for shipment723.

In some instances, quality control measures are taken. After errorcorrection, quality control steps include for example interaction with awafer having sequencing primers for amplification of the error correctedproduct 716, sealing the wafer to a chamber containing error correctedamplification product 717, and performing an additional round ofamplification 718. The nanoreactor is opened 719 and the products arepooled 720 and sequenced 721. After an acceptable quality controldetermination is made, the packaged product 722 is approved for shipment723.

In some instances, a nucleic acid generated by a workflow such as thatin FIG. 7 is subject to mutagenesis using overlapping primers disclosedherein. In some instances, a library of primers are generated by in situpreparation on a solid support and utilize single nucleotide extensionprocess to extend multiple oligomers in parallel. A deposition device,such as a material deposition device, is designed to release reagents ina step wise fashion such that multiple polynucleotides extend, inparallel, one residue at a time to generate oligomers with apredetermined nucleic acid sequence 702.

Computer Systems

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. In various instances, the methods and systems of thedisclosure may further comprise software programs on computer systemsand use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of thedisclosure. The computer systems may be programmed to interface betweenthe user specified base sequence and the position of a materialdeposition device to deliver the correct reagents to specified regionsof the substrate.

The computer system 800 illustrated in FIG. 8 may be understood as alogical apparatus that can read instructions from media 811 and/or anetwork port 805, which can optionally be connected to server 809 havingfixed media 812. The system, such as shown in FIG. 8 can include a CPU801, disk drives 803, optional input devices such as keyboard 815 and/ormouse 816 and optional monitor 807. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 822 as illustrated in FIG. 8.

As illustrated in FIG. 9, a high speed cache 904 can be connected to, orincorporated in, the processor 902 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 902. The processor 902 is connected to a north bridge 906 by aprocessor bus 908. The north bridge 906 is connected to random accessmemory (RAM) 910 by a memory bus 912 and manages access to the RAM 910by the processor 902. The north bridge 906 is also connected to a southbridge 914 by a chipset bus 916. The south bridge 914 is, in turn,connected to a peripheral bus 918. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 918. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip. In someinstances, system 900 can include an accelerator card 922 attached tothe peripheral bus 918. The accelerator can include field programmablegate arrays (FPGAs) or other hardware for accelerating certainprocessing. For example, an accelerator can be used for adaptive datarestructuring or to evaluate algebraic expressions used in extended setprocessing.

Software and data are stored in external storage 924 and can be loadedinto RAM 910 and/or cache 904 for use by the processor. The system 900includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample instances of the present disclosure. In this example, system 900also includes network interface cards (NICs) 920 and 921 connected tothe peripheral bus for providing network interfaces to external storage,such as Network Attached Storage (NAS) and other computer systems thatcan be used for distributed parallel processing.

FIG. 10 is a diagram showing a network 1000 with a plurality of computersystems 1002 a, and 1002 b, a plurality of cell phones and personal dataassistants 1002 c, and Network Attached Storage (NAS) 1004 a, and 1004b. In example instances, systems 1002 a, 1002 b, and 1002 c can managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 1004 a and 1004 b. A mathematical model can beused for the data and be evaluated using distributed parallel processingacross computer systems 1002 a, and 1002 b, and cell phone and personaldata assistant systems 1002 c. Computer systems 1002 a, and 1002 b, andcell phone and personal data assistant systems 1002 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 1004 a and 1004 b. FIG. 10 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various instances of thepresent disclosure. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface. In some example instances, processors can maintainseparate memory spaces and transmit data through network interfaces,back plane or other connectors for parallel processing by otherprocessors. In other instances, some or all of the processors can use ashared virtual address memory space.

FIG. 11 is a block diagram of a multiprocessor computer system using ashared virtual address memory space in accordance with an exampleinstance. The system includes a plurality of processors 1102 a-f thatcan access a shared memory subsystem 1104. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)1106 a-f in the memory subsystem 1104. Each MAP 1106 a-f can comprise amemory 1108 a-f and one or more field programmable gate arrays (FPGAs)1110 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 1110a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample instances. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 1108a-f, allowing it to execute tasks independently of, and asynchronouslyfrom the respective microprocessor 1102 a-f. In this configuration, aMAP can feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other instances, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 9, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 922 illustrated in FIG. 9.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of thedisclosure. Changes therein and other uses which are encompassed withinthe spirit of the disclosure as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200X spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A descum process was used to remove residualresist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 wattsfor 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary polynucleotide of 50 bp (“50-merpolynucleotide”) using polynucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 2.5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQID NO.: 2), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer.

TABLE 3 Synthesis protocols General DNA Synthesis Table 3 Process NameProcess Step Time (sec) WASH Acetonitrile System Flush 4 (AcetonitrileWash Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Incubate for 25sec 25 WASH AcetonitrileSystem Flush 4 (Acetonitrile Wash Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION ActivatorManifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 ActivatorFlow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25sec 25WASH Acetonitrile System Flush 4 (Acetonitrile Wash Flow) Acetonitrileto Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPINGCapA + B to Flowcell 15 (CapA + B, 1:1, Flow) WASH Acetonitrile SystemFlush 4 (Acetonitrile Wash Flow) Acetonitrile to Flowcell 15Acetonitrile System Flush 4 OXIDATION Oxidizer to Flowcell 18 (OxidizerFlow) WASH Acetonitrile System Flush 4 (Acetonitrile Wash Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 SystemFlush 4 Acetonitrile System Flush 4 DEBLOCKING Deblock to Flowcell 36(Deblock Flow) WASH Acetonitrile System Flush 4 (Acetonitrile Wash Flow)N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell18 N2 System Flush 4.13 Acetonitrile System Flush 4.13 Acetonitrile toFlowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After polynucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to recoverpolynucleotides. The recovered polynucleotides were then analyzed on aBioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an OligonucleotideSynthesis Device

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer polynucleotide(“100-mer polynucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotesThymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 3) on two different silicon chips, the first oneuniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the polynucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3; SEQ ID NO.: 4) and a reverse(5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO.: 5) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL polynucleotide extracted from the surface, and water up to50 uL) using the following thermal cycling program:

98° C., 30 sec

98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles

72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharppeaks at the 100-mer position. Next, the PCR amplified samples werecloned, and Sanger sequenced. Table 4 summarizes the results from theSanger sequencing for samples taken from spots 1-5 from chip 1 and forsamples taken from spots 6-10 from chip 2.

TABLE 4 Sequencing results Spot Error rate Cycle efficiency  1  1/763 bp99.87%  2  1/824 bp 99.88%  3  1/780 bp 99.87%  4  1/429 bp 99.77%  51/1525 bp 99.93%  6 1/1615 bp 99.94%  7  1/531 bp 99.81%  8 1/1769 bp99.94%  9  1/854 bp 99.88% 10 1/1451 bp 99.93%

Thus, the high quality and uniformity of the synthesized polynucleotideswere repeated on two chips with different surface chemistries. Overall,89% of the 100-mers that were sequenced were perfect sequences with noerrors, corresponding to 233 out of 262.

Table 5 summarizes error characteristics for the sequences obtained fromthe polynucleotide samples from spots 1-10.

TABLE 5 Error characteristics Sample ID/Spot no. OSA_0046/1 OSA_0047/2OSA_0048/3 OSA_0049/4 OSA_0050/5 Total 32 32 32 32 32 SequencesSequencing 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 Quality Oligo 23of 25 25 of 27 22 of 26 18 of 21 24 of 25 Quality ROI 2500 2698 25612122 2499 Match Count ROI 2 2 1 3 1 Mutation ROI Multi 0 0 0 0 0 BaseDeletion ROI Small 1 0 0 0 0 Insertion ROI 0 0 0 0 0 Single BaseDeletion Large 0 0 1 0 0 Deletion Count Mutation: 2 2 1 2 1 G > AMutation: 0 0 0 1 0 T > C ROI Error 3 2 2 3 1 Count ROI Error Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 834 in 1350 in 1282 in 708 in2500 ROI MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 Minus in763 in 824 in 780 in 429 in 1525 Primer Error Rate Sample ID/Spot no.OSA_0051/6 OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_0055/10 Total 32 32 3232 32 Sequences Sequencing 29 of 30 27 of 31 29 of 31 28 of 29 25 of 28Quality Oligo 25 of 29 22 of 27 28 of 29 26 of 28 20 of 25 Quality ROI2666 2625 2899 2798 2348 Match Count ROI 0 2 1 2 1 Mutation ROI Multi 00 0 0 0 Base Deletion ROI Small 0 0 0 0 0 Insertion ROI 0 0 0 0 0 SingleBase Deletion Large 1 1 0 0 0 Deletion Count Mutation: 0 2 1 2 1 G > AMutation: 0 0 0 0 0 T > C ROI Error 1 3 1 2 1 Count ROI Error Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 2667 in 876 in 2900 in 1400 in2349 ROI MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 Minus in1615 in 531 in 1769 in 854 in 1451 Primer Error Rate

Example 4: Design of Antibody Scaffolds

To generate scaffolds, structural analysis, repertoire sequencinganalysis of the heavy chain, and specific analysis of heterodimerhigh-throughput sequencing datasets were performed. Each heavy chain wasassociated with each light chain scaffold. Each heavy chain scaffold wasassigned 5 different long CDRH3 loop options. Each light chain scaffoldwas assigned 5 different L3 scaffolds. The heavy chain CDRH3 stems werechosen from the frequently observed long H3 loop stems (10 amino acidson the N-terminus and the C-terminus) found both across individuals andacross V-gene segments. The light chain scaffold L3s were chosen fromheterodimers comprising long H3s. Direct heterodimers based oninformation from the Protein Data Bank (PDB) and deep sequencingdatasets were used in which CDR H1, H2, L1, L2, L3, and CDRH3 stems werefixed. The various scaffolds were then formatted for display on phage toassess for expression.

Structural Analysis

About 2,017 antibody structures were analyzed from which 22 structureswith long CDRH3s of at least 25 amino acids in length were observed. Theheavy chains included the following: IGHV1-69, IGHV3-30, IGHV4-49, andIGHV3-21. The light chains identified included the following: IGLV3-21,IGKV3-11, IGKV2-28, IGKV1-5, IGLV1-51, IGLV1-44, and IGKV1-13. In theanalysis, four heterodimer combinations were observed multiple timesincluding: IGHV4-59/61-IGLV3-21, IGHV3-21-IGKV2-28, IGHV1-69-IGKV3-11,and IGHV1-69-IGKV1-5. An analysis of sequences and structures identifiedintra-CDRH3 disulfide bonds in a few structures with packing of bulkyside chains such as tyrosine in the stem providing support for long H3stability. Secondary structures including beta-turn-beta sheets and a“hammerhead” subdomain were also observed.

Repertoire Analysis

A repertoire analysis was performed on 1,083,875 IgM+/CD27-naïve B cellreceptor (BCR) sequences and 1,433,011 CD27+ sequences obtained byunbiased 5′RACE from 12 healthy controls. The 12 healthy controlscomprised equal numbers of male and female and were made up of 4Caucasian, 4 Asian, and 4 Hispanic individuals. The repertoire analysisdemonstrated that less than 1% of the human repertoire comprises BCRswith CDRH3s longer than 21 amino acids. A V-gene bias was observed inthe long CDR3 subrepertoire, with IGHV1-69, IGHV4-34, IGHV1-18, andIGHV1-8 showing preferential enrichment in BCRs with long H3 loops. Abias against long loops was observed for IGHV3-23, IGHV4-59/61,IGHV5-51, IGHV3-48, IGHV3-53/66, IGHV3-15, IGHV3-74, IGHV3-73, IGHV3-72,and IGHV2-70. The IGHV4-34 scaffold was demonstrated to be autoreactiveand had a short half-life.

Viable N-terminal and C-terminal CDRH3 scaffold variation for long loopswere also designed based on the 5′RACE reference repertoire. About81,065 CDRH3s of amino acid length 22 amino acids or greater wereobserved. By comparing across V-gene scaffolds, scaffold-specific H3stem variation was avoided as to allow the scaffold diversity to becloned into multiple scaffold references.

Heterodimer Analysis

Heterodimer analysis was performed on scaffolds and variant sequencesand lengths of the scaffolds were assayed.

Structural Analysis

Structural analysis was performed using GPCR scaffolds of variantsequences and lengths were assayed.

Example 5: Generation of GPCR Antibody Libraries

Based on GPCR-ligand interaction surfaces and scaffold arrangements,libraries were designed and de novo synthesized. See Example 4.Referring to FIG. 5, 10 variant sequences were designed for the variabledomain, heavy chain 503, 237 variant sequences were designed for theheavy chain complementarity determining region 3 507, and 44 variantsequences were designed for the variable domain, light chain 513. Thefragments were synthesized as three fragments as seen in FIG. 6following similar methods as described in Examples 1-3.

Following de novo synthesis, 10 variant sequences were generated for thevariable domain, heavy chain 602, 236 variant sequences were generatedfor the heavy chain complementarity determining region 3 604, and 43variant sequences were designed for a region comprising the variabledomain 606, light chain and CDRL3 and of which 9 variants for variabledomain, light chain were designed. This resulted in a library with about10⁵ diversity (10×236×43). This was confirmed using next generationsequencing (NGS) with 16 million reads. The normalized sequencing readsfor each of the 10 variants for the variable domain, heavy chain wasabout 1. The normalized sequencing reads for each of the 43 variants forthe variable domain, light chain was about 1. The normalized sequencingreads for 236 variant sequences for the heavy chain complementaritydetermining region 3 were about 1.

The various light and heavy chains were then tested for expression andprotein folding. The 10 variant sequences for variable domain, heavychain included the following: IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21,IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, and IGHV4-59/61.Of the 10 variant sequences, IGHV1-18, IGHV1-69, and IGHV3-30/33rnexhibited improved characteristics such as improved thermostability. 9variant sequences for variable domain, light chain included thefollowing: IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20,IGKV4-1, IGLV1-51, and IGLV2-14. Of the 9 variant sequences, IGKV1-39,IGKV3-15, IGLV1-51, and IGLV2-14 exhibited improved characteristics suchas improved thermostability.

Example 6: Expression of GPCR Antibody Libraries in HEK293 Cells

Following generation of GPCR antibody libraries, about 47 GPCRs wereselected for screening. GPCR constructs about 1.8 kb to about 4.5 kb insize were designed in a pCDNA3.1 vector. The GPCR constructs were thensynthesized following similar methods as described in Examples 4-5including hierarchal assembly. Of the 47 GPCR constructs, 46 GPCRconstructs were synthesized.

The synthesized GPCR constructs were transfected in HEK293 and assayedfor expression using immunofluorescence. HEK293 cells were transfectedwith the GPCR constructs comprising an N-terminally hemagglutinin(HA)-tagged human Y₁ receptor (data not shown). Following 24-48 hours oftransfection, cells were washed with phosphate buffered saline (PBS) andfixed with 4% paraformaldehyde. Cells were stained using fluorescentprimary antibody directed towards the HA tag or secondary antibodiescomprising a fluorophore and DAPI to visualize the nuclei in blue. HumanY₁ receptor was visualized on the cell surface in non-permeabilizedcells and on the cell surface and intracellularly in permeabilized cells(data not shown).

GPCR constructs were also visualized by designing GPCR constructscomprising auto-fluorescent proteins. Human Y₁ receptor comprised EYFPfused to its C-terminus, and human Y₅ receptor comprised ECFP fused toits C-terminus (data not shown). HEK293 cells were transfected withhuman Y₁ receptor or co-transfected with human Y₁ receptor and human Y₅receptor. Following transfection cells were washed and fixed with 4%paraformaldehyde. Cells were stained with DAPI. Localization of human Y₁receptor and human Y₅ receptor were visualized by fluorescencemicroscopy.

Example 7 Design of Immunoglobulin Library

An immunoglobulin scaffold library was designed for placement of GPCRbinding domains and for improving stability for a range of GPCR bindingdomain encoding sequences. The immunoglobulin scaffold included a VHdomain attached with a VL domain with a linker. Variant nucleic acidsequences were generated for the framework elements and CDR elements ofthe VH domain and VL domain. The structure of the design is shown inFIG. 12A. A full domain architecture is shown in FIG. 12B. Sequences forthe leader, linker, and pIII are listed in Table 7.

TABLE 7 Nucleotide sequences SEQ ID NO Domain Sequence 6 LeaderGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGG CCATGGCC 7 LinkerGCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTG GCGGTGGCGGATCGCATGCATCC 8 pIIICGCGCGGCCGCTGGAAGCGGCTCCCACCATCACCATC ACCAT

The VL domains that were designed include IGKV1-39, IGKV3-15, IGLV1-51,and IGLV2-14. Each of four VL domains were assembled with theirrespective invariant four framework elements (FW1, FW2, FW3, FW4) andvariable 3 CDR (L1, L2, L3) elements. For IGKV1-39, there was 490variants designed for L1, 420 variants designed for L2, and 824 variantsdesigned for L3 resulting in a diversity of 1.7×10⁸ (490*420*824). ForIGKV3-15, there was 490 variants designed for L1, 265 variants designedfor L2, and 907 variants designed for L3 resulting in a diversity of1.2×10⁸ (490*265*907). For IGLV1-51, there was 184 variants designed forL1, 151 variants designed for L2, and 824 variants designed for L3resulting in a diversity of 2.3×10⁷ (184*151*824). IGLV2-14, 967variants designed for L1, 535 variants designed for L2, and 922 variantsdesigned for L3 resulting in a diversity of 4.8 10⁸ (967*535*922). Table8 lists the amino acid sequences and nucleotide sequences for the fourframework elements (FW1, FW2, FW3, FW4) for IGLV1-51. Table 9 lists thevariable 3 CDR (L1, L2, L3) elements for IGLV1-51. Variant amino acidsequences and nucleotide sequences for the four framework elements (FW1,FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements were alsodesigned for IGKV1-39, IGKV3-15, and IGLV2-14.

TABLE 8 Sequences for IGLV1-51 framework elements Element SEQ ID NOAmino Acid Sequence SEQ ID NO Nucleotide Sequence FW1  9QSVLTQPPSVSAAPGQKVTISC 10 CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCA TCTCCTGC FW2 11 WYQQLPGTAPKLLIY 12TGGTATCAGCAGCTCCCAGGAACAGCCCC CAAACTCCTCATTTAT FW3 13GIPDRFSGSKSGTSATLGITGL 14 GGGATTCCTGACCGATTCTCTGGCTCCAA QTGDEADYYGTCTGGCACGTCAGCCACCCTGGGCATCA CCGGACTCCAGACTGGGGACGAGGCCGAT TATTAC FW415 GGGTKLTVL 16 GGCGGAGGGACCAAGCTGACCGTCCTA

TABLE 9 Sequences for IGLV1-51 CDR elements SEQ SEQ ID NOAmino Acid Sequence ID NO Nucleotide Sequence IGLV1-51-L1   17SGSSSNIGSNHVS  200 TCTGGAAGCAGCTCCAACATTGGGAGTAATCATGTATCC   18SGSSSNIGNNYLS  201 TCTGGAAGCAGCTCCAACATTGGGAATAATTATCTATCC   19SGSSSNIANNYVS  202 TCTGGAAGCAGCTCCAACATTGCGAATAATTATGTATCC   20SGSSPNIGNNYVS  203 TCTGGAAGCAGCCCCAACATTGGGAATAATTATGTATCG   21SGSRSNIGSNYVS  204 TCTGGAAGCAGATCCAATATTGGGAGTAATTATGTTTCG   22SGSSSNVGDNYVS  205 TCTGGAAGCAGCTCCAACGTTGGCGATAATTATGTTTCC   23SGSSSNIGIQYVS  206 TCTGGAAGCAGCTCCAACATTGGGATTCAATATGTATCC   24SGSSSNVGNNFVS  207 TCTGGAAGCAGCTCCAATGTTGGTAACAATTTTGTCTCC   25SGSASNIGNNYVS  208 TCTGGAAGCGCCTCCAACATTGGGAATAATTATGTATCC   26SGSGSNIGNNDVS  209 TCTGGAAGCGGCTCCAATATTGGGAATAATGATGTGTCC   27SGSISNIGNNYVS  210 TCTGGAAGCATCTCCAACATTGGTAATAATTATGTATCC   28SGSISNIGKNYVS  211 TCTGGAAGCATCTCCAACATTGGGAAAAATTATGTGTCG   29SGSSSNIGHNYVS  212 TCTGGAAGCAGCTCCAACATTGGGCATAATTATGTATCG   30PGSSSNIGNNYVS  213 CCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC   31SGSTSNIGIHYVS  214 TCTGGAAGCACCTCCAACATTGGAATTCATTATGTATCC   32SGSSSNIGSHYVS  215 TCTGGAAGCAGCTCCAACATTGGCAGTCATTATGTTTCC   33SGSSSNIGNEYVS  216 TCCGGAAGCAGCTCCAACATTGGAAATGAATATGTATCC   34SGSTSNIGNNYIS  217 TCTGGAAGCACCTCCAACATTGGAAATAATTATATATCG   35SGSSSNIGNHFVS  218 TCTGGAAGCAGCTCCAATATTGGGAATCATTTTGTATCG   36SGSSSNIGNNYVA  219 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTGGCC   37SGSSSNIGSYYVS  220 TCTGGAAGCAGCTCCAACATTGGAAGTTATTATGTATCC   38SGSGFNIGNNYVS  221 TCTGGAAGTGGTTTCAACATTGGGAATAATTATGTCTCT   39SGSTSNIGNNYVS  222 TCTGGAAGCACCTCCAACATTGGGAATAATTATGTGTCC   40SGSSSDIGNNYVS  223 TCTGGAAGCAGCTCCGACATTGGCAATAATTATGTATCC   41SGSSSNIGNNVVS  224 TCTGGAAGCAGCTCCAACATTGGGAATAATGTTGTATCC   42SGSKSNIGKNYVS  225 TCTGGAAGCAAGTCTAACATTGGGAAAAATTATGTATCC   43SGSSTNIGNNYVS  226 TCTGGAAGCAGCACCAACATTGGGAATAATTATGTATCC   44SGSISNIGDNYVS  227 TCTGGAAGCATCTCCAACATTGGGGATAATTATGTATCC   45SGSSSNIGSKDVS  228 TCTGGAAGCAGCTCCAACATTGGGAGTAAGGATGTATCA   46SGSSSNIENNDVS  229 TCTGGAAGCAGCTCCAACATTGAGAATAATGATGTATCG   47SGSSSNIGNHYVS  230 TCTGGAAGCAGCTCCAACATTGGGAATCATTATGTATCC   48SGSSSNIGKDFVS  231 TCTGGAAGCAGCTCCAACATTGGGAAGGATTTTGTCTCC   49SGSTSNIGSNFVS  232 TCTGGCAGTACTTCCAACATCGGAAGTAATTTTGTTTCC   50SGSTSNIGHNYVS  233 TCTGGAAGCACCTCCAACATTGGGCATAATTATGTATCC   51SASSSNIGNNYVS  234 TCTGCAAGCAGCTCCAACATTGGGAATAATTATGTATCC   52SGSSSSIGNNYVS  235 TCTGGAAGCAGCTCCAGCATTGGCAATAATTATGTATCC   53SGSSSTIGNNYVS  236 TCTGGAAGCAGCTCCACCATTGGGAATAATTATGTATCC   54SGSSSNIENNYVS  237 TCTGGAAGCAGCTCCAACATTGAAAATAATTATGTATCC   55SGSSSNIGNQYVS  238 TCTGGAAGCAGCTCCAACATTGGGAATCAGTATGTATCC   56SGSSSNIGNNYVF  239 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATTC   57SGSSSNIGRNYVS  240 TCTGGAAGCAGCTCCAACATTGGGAGGAATTATGTCTCC   58SGGSSNIGNYYVS  241 TCTGGAGGCAGCTCCAACATTGGAAATTATTATGTATCG   59SGSSSNIGDNYVS  242 TCTGGAAGCAGCTCCAACATTGGAGATAATTATGTCTCC   60SGGSSNIGINYVS  243 TCTGGAGGCAGCTCCAACATTGGAATTAATTATGTATCC   61SGGSSNIGKNYVS  244 TCTGGAGGCAGCTCCAACATTGGGAAGAATTATGTATCC   62SGSSSNIGKRSVS  245 TCTGGAAGCAGCTCCAACATTGGGAAGAGATCTGTATCG   63SGSRSNIGNNYVS  246 TCTGGAAGCAGATCCAACATTGGGAATAACTATGTATCC   64SGSSSNIGNNLVS  247 TCGGGAAGCAGCTCCAACATTGGGAATAATCTTGTTTCC   65SGSSSNIGINYVS  248 TCTGGAAGCAGCTCCAACATTGGGATCAATTATGTATCC   66SGSSSNIGNNFVS  249 TCTGGAAGCAGCTCCAACATCGGGAATAATTTTGTATCC   67SGTSSNIGRNFVS  250 TCTGGAACCAGCTCCAACATTGGCAGAAATTTTGTATCC   68SGRRSNIGNNYVS  251 TCTGGAAGGAGGTCCAACATTGGAAATAATTATGTGTCC   69SGGSFNIGNNYVS  252 TCTGGAGGCAGCTTCAATATTGGGAATAATTATGTATCC   70SGSTSNIGENYVS  253 TCTGGAAGCACTTCCAACATTGGGGAGAATTATGTGTCC   71SGSSSNIGSDYVS  254 TCTGGAAGCAGCTCCAATATTGGGAGTGATTATGTATCC   72SGTSSNIGSNYVS  255 TCTGGAACCAGCTCCAACATTGGGAGTAATTATGTATCC   73SGSSSNIGTNFVS  256 TCTGGAAGCAGCTCCAACATTGGGACTAATTTTGTATCC   74SGSSSNFGNNYVS  257 TCTGGAAGCAGCTCCAACTTTGGGAATAATTATGTATCC   75SGSTSNIGNNHVS  258 TCTGGAAGCACCTCCAACATTGGGAATAATCATGTATCC   76SGSSSNIGNDFVS  259 TCTGGAAGCAGCTCCAACATTGGGAATGATTTTGTATCC   77SGSSSDIGDNYVS  260 TCTGGAAGCAGCTCCGACATTGGCGATAATTATGTGTCC   78SGSSSNIGKYYVS  261 TCTGGAAGCAGCTCCAACATTGGGAAATATTATGTATCC   79SGSSSNIGGNYVS  262 TCTGGAAGCAGCTCCAACATTGGCGGTAATTATGTATCC   80SGSSSNTGNNYVS  263 TCTGGAAGCAGCTCCAACACTGGGAATAATTATGTATCC   81SGSSSNVGNNYVS  264 TCTGGAAGCAGCTCCAACGTTGGGAATAATTATGTGTCT   82SGSSSNIANNFVS  265 TCTGGAAGCAGCTCCAACATTGCGAATAATTTTGTATCC   83SGSSSNIGNDYVS  266 TCTGGAAGCAGCTCCAACATTGGGAATGATTATGTATCC   84SGSTSNIENNYVS  267 TCTGGAAGCACCTCCAATATTGAGAATAATTATGTTTCC   85SGGSSNIGNNDVS  268 TCTGGAGGCAGCTCCAATATTGGCAATAATGATGTGTCC   86SGSTSNIGNHYVS  269 TCTGGAAGCACCTCCAACATTGGGAATCATTATGTATCC   87SGSSSNIGDNDVS  270 TCAGGAAGCAGCTCCAATATTGGGGATAATGATGTATCC   88SGYSSNIGNNYVS  271 TCTGGATACAGCTCCAACATTGGGAATAATTATGTATCC   89SGSGSNIGNNFVS  272 TCTGGAAGCGGCTCCAACATTGGAAATAATTTTGTATCC   90SGSSSNIWNNYVS  273 TCTGGAAGCAGCTCCAACATTTGGAATAATTATGTATCC   91FGSSSNIGNNYVS  274 TTTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC   92SGSSSNIEKNYVS  275 TCTGGAAGCAGCTCCAACATTGAGAAGAATTATGTATCC   93SGSRSNIGNYYVS  276 TCTGGAAGTAGATCCAATATTGGAAATTATTATGTATCC   94SGTKSNIGNNYVS  277 TCTGGAACCAAGTCAAACATTGGGAATAATTATGTATCT   95SGSTSNIGNYYVS  278 TCTGGAAGCACCTCCAACATTGGGAATTATTATGTATCC   96SGTSSNIGNNYVA  279 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTGGCC   97PGTSSNIGNNYVS  280 CCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC   98SGSTSNIGINYVS  281 TCCGGAAGCACCTCCAACATTGGGATTAATTATGTATCC   99SGSSSNIGSNLVS  282 TCTGGAAGCAGCTCCAACATTGGGAGTAATCTGGTATCC  100SGSSSNIENNHVS  283 TCTGGAAGCAGCTCCAACATTGAGAATAATCATGTATCC  101SGTRSNIGNNYVS  284 TCTGGAACCAGGTCCAACATCGGCAATAATTATGTTTCG  102SGSTSNIGDNYVS  285 TCTGGAAGCACCTCCAACATTGGGGACAATTATGTTTCC  103SGGSSNIGKNFVS  286 TCTGGAGGCAGTTCCAACATTGGGAAGAATTTTGTATCC  104SGSRSDIGNNYVS  287 TCTGGAAGCAGGTCCGACATTGGGAATAATTATGTATCC  105SGTSSNIGNNDVS  288 TCTGGAACTAGCTCCAACATTGGGAATAATGATGTATCC  106SGSSSNIGSKYVS  289 TCTGGAAGCAGCTCCAACATTGGGAGTAAATATGTATCA  107SGSSFNIGNNYVS  290 TCTGGAAGCAGCTTCAACATTGGGAATAATTATGTATCC  108SGSSSNIGNTYVS  291 TCTGGAAGCAGCTCCAACATTGGGAATACTTATGTATCC  109SGSSSNIGDNHVS  292 TCTGGAAGCAGCTCCAATATTGGGGATAATCATGTATCC  110SGSSSNIGNNHVS  293 TCTGGAAGCAGCTCCAACATTGGCAATAATCATGTTTCC  111SGSTSNIGNNDVS  294 TCTGGAAGCACCTCCAACATTGGGAATAATGATGTATCC  112SGSRSNVGNNYVS  295 TCTGGAAGCAGATCCAACGTTGGCAATAATTATGTTTCA  113SGGTSNIGKNYVS  296 TCCGGAGGCACCTCCAACATTGGGAAGAATTATGTGTCT  114SGSSSNIADNYVS  297 TCTGGAAGCAGCTCCAACATTGCCGATAATTATGTTTCC  115SGSSSNIGANYVS  298 TCTGGAAGCAGCTCCAACATTGGCGCCAATTATGTATCC  116SGSSSNIGSNYVA  299 TCTGGAAGCAGCTCCAACATTGGGAGTAATTATGTGGCC  117SGSSSNIGNNFLS  300 TCTGGAAGCAGCTCCAACATTGGGAACAATTTTCTCTCC  118SGRSSNIGKNYVS  301 TCTGGAAGAAGCTCCAACATTGGGAAGAATTATGTATCC  119SGSSPNIGANYVS  302 TCTGGAAGCAGCCCCAACATTGGGGCTAATTATGTATCC  120SGSSSNIGPNYVS  303 TCCGGAAGCAGCTCCAACATTGGGCCTAATTATGTGTCC  121SGSSSTIGNNYIS  304 TCTGGAAGCAGCTCCACCATTGGGAATAATTATATATCC  122SGSSSNIGNYFVS  305 TCTGGAAGCAGCTCCAACATTGGGAATTATTTTGTATCC  123SGSRSNIGNNFVS  306 TCTGGAAGCCGCTCCAACATTGGTAATAATTTTGTATCC  124SGGSSNIGSNFVS  307 TCTGGAGGCAGCTCCAACATTGGGAGTAATTTTGTATCC  125SGSSSNIGYNYVS  308 TCTGGAAGCAGCTCCAACATTGGGTATAATTATGTATCC  126SGTSSNIENNYVS  309 TCTGGAACCAGCTCGAACATTGAGAACAATTATGTATCC  127SGSSSNIGNYYVS  310 TCTGGAAGTAGCTCCAACATTGGGAATTATTATGTATCC  128SGSTSNIGKNYVS  311 TCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCC  129SGSSSNIGTYYVS  312 TCTGGAAGCAGTTCCAACATTGGGACTTATTATGTCTCT  130SGSSSNVGKNYVS  313 TCTGGAAGCAGCTCCAACGTTGGGAAAAATTATGTATCT  131SGSTSNIGDNFVS  314 TCTGGAAGCACCTCCAACATTGGGGATAATTTTGTATCC  132SGSTSNIGTNYVS  315 TCTGGAAGCACCTCCAACATTGGAACTAATTATGTTTCC  133SGGTSNIGNNYVS  316 TCTGGAGGTACTTCCAACATTGGGAATAATTATGTCTCC  134SGSYSNIGNNYVS  317 TCTGGAAGCTACTCCAATATTGGGAATAATTATGTATCC  135SGSSSNIEDNYVS  318 TCTGGAAGCAGCTCCAACATTGAAGATAATTATGTATCC  136SGSSSNIGKHYVS  319 TCTGGAAGCAGCTCCAACATTGGGAAACATTATGTATCC  137SGSGSNIGSNYVS  320 TCCGGTTCCGGCTCAAACATTGGAAGTAATTATGTCTCC  138SGSSSNIGNNYIS  321 TCTGGAAGCAGCTCCAACATTGGAAATAATTATATATCA  139SGASSNIGNNYVS  322 TCTGGAGCCAGTTCCAACATTGGGAATAATTATGTTTCC  140SGRTSNIGNNYVS  323 TCTGGACGCACCTCCAACATCGGGAACAATTATGTATCC  141SGGSSNIGSNYVS  324 TCTGGAGGCAGCTCCAATATTGGGAGTAATTACGTATCC  142SGSGSNIGNNYVS  325 TCTGGAAGCGGCTCCAACATTGGGAATAATTATGTATCC  143SGSTSNIGSNYVS  326 TCTGGAAGCACCTCCAACATTGGGAGTAATTATGTATCC  144SGSSSSIGNNYVA  327 TCTGGAAGCAGCTCCAGCATTGGGAATAATTATGTGGCG  145SGSSSNLGNNYVS  328 TCTGGAAGCAGTTCCAACCTTGGAAATAATTATGTATCC  146SGTSSNIGKNYVS  329 TCTGGAACCAGCTCCAACATTGGGAAAAATTATGTATCC  147SGSSSDIGNKYIS  330 TCTGGAAGCAGCTCCGATATTGGGAACAAGTATATATCC  148SGSSSNIGSNYIS  331 TCTGGAAGCAGCTCCAACATTGGAAGTAATTACATATCC  149SGSTSNIGANYVS  332 TCTGGAAGCACCTCCAACATTGGGGCTAACTATGTGTCC  150SGSSSNIGNKYVS  333 TCTGGAAGCAGCTCCAACATTGGGAATAAGTATGTATCC  151SGSSSNIGNNYGS  334 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGGATCC  152SGSTSNIANNYVS  335 TCTGGAAGCACCTCCAACATTGCGAATAATTATGTATCC  153SGSYSNIGSNYVS  336 TCTGGAAGCTACTCCAATATTGGGAGTAATTATGTATCC  154SGSSSNIGSNFVS  337 TCTGGAAGCAGCTCCAACATTGGGAGTAATTTTGTATCC  155SGSSSNLENNYVS  338 TCTGGAAGCAGCTCCAATCTTGAGAATAATTATGTATCC  156SGSISNIGSNYVS  339 TCTGGAAGCATCTCCAATATTGGCAGTAATTATGTATCC  157SGSSSDIGSNYVS  340 TCTGGAAGCAGCTCCGACATTGGGAGTAATTATGTATCC  158SGSSSNIGTNYVS  341 TCTGGAAGCAGCTCCAACATTGGGACTAATTATGTATCC  159SGSSSNIGKNFVS  342 TCTGGAAGCAGCTCCAACATTGGGAAGAATTTTGTATCC  160SGSSSNIGNNFIS  343 TCTGGAAGCAGCTCCAACATTGGGAATAATTTTATATCC  161SGGSSNIGNNYVS  344 TCTGGAGGCAGCTCCAACATTGGCAATAATTATGTTTCC  162SGSSSNIGENYVS  345 TCTGGAAGCAGCTCCAACATTGGGGAGAATTATGTATCC  163SGSSSNIGNNFVA  346 TCTGGAAGCAGCTCCAATATTGGGAATAATTTTGTGGCC  164SGGSSNIGNNYVA  347 TCTGGAGGCAGCTCCAACATTGGGAATAATTATGTAGCC  165SGSSSHIGNNYVS  348 TCTGGAAGCAGCTCCCACATTGGAAATAATTATGTATCC  166SGSSSNIGSNDVS  349 TCTGGAAGCAGCTCCAATATTGGAAGTAATGATGTATCG  167SGSSSNIGNNYVT  350 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTAACC  168SGSSSNIGNNPVS  351 TCTGGAAGCAGCTCCAACATTGGGAATAATCCTGTATCC  169SGGSSNIGNHYVS  352 TCTGGAGGCAGCTCCAATATTGGGAATCATTATGTATCC  170SGTSSNIGNNYVS  353 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC  171SGSSSNIGSNYVS  354 TCTGGAAGCAGCTCCAACATTGGAAGTAATTATGTCTCG  172SGGTSNIGSNYVS  355 TCTGGAGGCACCTCCAACATTGGAAGTAATTATGTATCC  173SGSKSNIGNNYVS  356 TCTGGAAGCAAGTCCAACATTGGGAATAATTATGTATCC  174SGRSSNIGNNYVS  357 TCTGGAAGAAGCTCCAACATTGGGAATAATTATGTATCG  175SGSSSNVGSNYVS  358 TCTGGAAGCAGCTCCAACGTTGGGAGTAATTATGTTTCC  176SGSTSNIGNNFVS  359 TCTGGAAGCACCTCCAATATTGGGAATAATTTTGTATCC  177SGSNFNIGNNYVS  360 TCTGGAAGCAACTTCAACATTGGGAATAATTATGTCTCC  178SGSTSNIGYNYVS  361 TCTGGAAGCACCTCCAATATTGGATATAATTATGTATCC  179SGSSSNIVSNYVS  362 TCTGGAAGCAGCTCCAATATTGTAAGTAATTATGTATCC  180SGTSSNIGNNFVS  363 TCTGGAACCAGCTCCAACATTGGGAATAATTTTGTATCC  181SGSSSNIGRNFVS  364 TCTGGAAGCAGCTCCAACATTGGGAGGAATTTTGTGTCC  182SGTTSNIGNNYVS  365 TCTGGAACGACCTCCAACATTGGGAATAATTATGTCTCC  183SGSSSNIGNNDVS  366 TCTGGAAGCAGCTCCAACATTGGGAATAATGATGTATCC  184SGSSSNIGNHDVS  367 TCTGGAAGCAGCTCCAACATTGGGAATCATGATGTATCC  185SGSSSNIGSSHVS  368 TCTGGAAGCAGCTCCAACATTGGAAGTAGTCATGTATCC  186SGSSSNIGIHYVS  369 TCTGGAAGCAGCTCCAACATTGGGATTCATTATGTATCC  187SGGGSNIGYNYVS  370 TCTGGAGGCGGCTCCAACATTGGCTATAATTATGTCTCC  188SGSSSNIGDHYVS  371 TCTGGAAGCAGCTCCAACATTGGGGATCATTATGTGTCG  189SGSSSNLGKNYVS  372 TCTGGAAGCAGCTCCAACCTTGGGAAGAATTATGTATCT  190SGSSSNIGDNFVS  373 TCTGGAAGCAGCTCCAACATTGGCGATAATTTTGTATCC  191SGSTSN1EKNYVS  374 TCTGGAAGCACCTCCAACATTGAGAAAAACTATGTATCG  192SGSSSNIGKDYVS  375 TCTGGAAGCAGCTCCAACATTGGGAAGGATTATGTATCC  193SGSSSNIGKNYVS  376 TCTGGAAGCAGCTCCAACATTGGGAAGAATTATGTATCC  194SGSSSNIGNNYVS  377 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  195SGSSSNIGNNYAS  378 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGCCTCC  196SGISSNIGNNYVS  379 TCTGGAATCAGCTCCAACATTGGGAATAATTATGTATCC  197TGSSSNIGNNYVS  380 ACTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  198SGTSSNIGNNHVS  381 TCTGGAACCAGCTCCAACATTGGGAATAATCATGTTTCC  199SGSRSNIGKNYVS  382 TCTGGAAGTCGTTCCAACATTGGGAAAAATTATGTATCC IGLV1-51-L2 383 DNNKRPP  534 GACAATAATAAGCGACCCCCA  384 ENNRRPS  535GAGAATAATAGGCGACCCTCA  385 DNNKQPS  536 GACAATAATAAGCAACCCTCA  386DNNKRPL  537 GACAATAACAAGCGACCCTTG  387 DNDKRPA  538GACAATGATAAGCGACCCGCA  388 DNHERPS  539 GACAATCATGAGCGACCCTCA  389ENRKRPS  540 GAAAACCGTAAGCGACCCTCA  390 DNDQRPS  541GACAATGATCAGCGACCCTCA  391 ENYKRPS  542 GAGAATTATAAGCGACCCTCA  392ENTKRPS  543 GAAAATACTAAGCGACCCTCA  393 DTEKRPS  544GACACTGAGAAGAGGCCCTCA  394 DNDKRPP  545 GACAATGATAAGCGACCCCCA  395DHNKRPS  546 GACCATAATAAGCGACCCTCA  396 GNNERPS  547GGCAATAATGAGCGACCCTCA  397 DTSKRPS  548 GACACTAGTAAGCGACCCTCA  398EYNKRPS  549 GAATATAATAAGCGCCCCTCA  399 ENIKRPS  550GAAAATATTAAGCGACCCTCA  400 DNVKRPS  551 GACAATGTTAAGCGACCCTCA  401ENDKRSS  552 GAAAACGATAAACGATCCTCA  402 ENNKRHS  553GAAAATAATAAGCGACACTCA  403 GNDQRPS  554 GGAAATGATCAGCGACCCTCA  404DNDRRPS  555 GACAATGATAGGCGACCCTCA  405 DNHKRPS  556GACAATCATAAGCGGCCCTCA  406 DNNDRPS  557 GACAATAATGACCGACCCTCA  407ENNQRPS  558 GAGAATAATCAGCGACCCTCA  408 DNNQRPS  559GACAATAATCAGCGACCCTCA  409 ENVKRPS  560 GAGAATGTTAAGCGACCCTCA  410DTYKRPS  561 GACACTTATAAGAGACCCTCA  411 NNNNRPS  562AACAATAATAACCGACCCTCA  412 GNNNRPS  563 GGCAATAATAATCGACCCTCA  413ENDQRPS  564 GAAAATGATCAGCGACCCTCA  414 DNNKRAS  565GACAATAATAAGCGAGCCTCA  415 DNDKRPL  566 GACAATGATAAGCGACCCTTA  416DTDERPS  567 GACACTGATGAGCGACCTTCA  417 DNRKRPS  568GACAATAGGAAGCGACCCTCA  418 DNDARPS  569 GACAATGATGCTCGACCCTCA  419DNNKRLS  570 GACAATAATAAGCGACTCTCA  420 DNDKRAS  571GACAATGATAAGCGAGCCTCA  421 DNTERPS  572 GACAATACTGAGCGACCCTCA  422DNNIRPS  573 GACAATAATATTCGACCCTCA  423 DNKRRPS  574GACAATAAGAGGCGACCCTCA  424 DDNNRPS  575 GACGATAATAACCGACCCTCA  425ANNRRPS  576 GCGAATAATCGACGACCCTCA  426 DNDKRLS  577GACAATGATAAGCGACTGTCA  427 DNNKRPA  578 GACAATAATAAGCGACCCGCA  428DNYRRPS  579 GACAATTATAGACGTCCCTCA  429 ANDQRPS  580GCCAATGATCAGCGACCCTCA  430 DNDKRRS  581 GACAATGATAAGCGACGCTCA  431DKNERPS  582 GACAAGAATGAGCGACCCTCA  432 DNKERPS  583GACAATAAGGAGCGACCCTCA  433 DNNKGPS  584 GACAATAATAAGGGACCCTCA  434ENDRRPS  585 GAAAATGATAGACGACCCTCA  435 ENDERPS  586GAAAATGATGAGCGACCCTCA  436 QNNKRPS  587 CAAAATAATAAGCGACCCTCA  437DNRERPS  588 GACAATCGTGAGCGACCCTCA  438 DNNRRPS  589GACAATAATAGACGACCCTCA  439 GNNRRPS  590 GGAAATAATAGGCGACCCTCA  440DNDNRPS  591 GACAATGATAACCGACCCTCA  441 EDNKRPS  592GAAGATAATAAGCGACCCTCA  442 DDDERPS  593 GACGATGATGAGCGGCCCTCA  443ASNKRPS  594 GCAAGTAATAAGCGACCCTCA  444 DNNKRSS  595GACAATAATAAGCGATCCTCA  445 QNNERPS  596 CAAAATAATGAGCGACCCTCA  446DDDRRPS  597 GACGATGATAGGCGACCCTCA  447 NNDKRPS  598AACAATGATAAGCGACCCTCA  448 DNNNRPS  599 GACAATAATAACCGACCCTCA  449DNNVRPS  600 GACAATAATGTGCGACCCTCA  450 ENNERPS  601GAAAATAATGAGCGACCCTCA  451 DNNHRPS  602 GACAATAATCACCGACCCTCA  452DNDERPS  603 GACAATGATGAGCGCCCCTCG  453 DNIRRPS  604GACAATATCCGGCGACCCTCA  454 DFNKRPS  605 GACTTTAATAAGCGACCCTCA  455ETNKRPS  606 GAAACTAATAAGCGACCCTCA  456 NDNKRPS  607AACGATAATAAGCGACCCTCA  457 DDNKRPS  608 GACGATAATAAGCGACCCTCA  458DNYKRPS  609 GACAATTATAAGCGACCCTCA  459 HNNKRPS  610CACAATAATAAGCGACCCTCA  460 DNHQRPS  611 GACAATCATCAGCGACCCTCA  461DNYKRAS  612 GACAATTATAAGCGAGCCTCA  462 DNIKRPS  613GACAATATTAAGCGACCCTCA  463 DTHKRPS  614 GACACTCATAAGCGACCCTCA  464DTNRRPS  615 GACACTAATAGGCGACCCTCT  465 DTNQRPS  616GACACTAATCAGCGACCCTCA  466 ESDKRPS  617 GAAAGTGATAAGCGACCCTCA  467DNDKRSS  618 GACAATGATAAGCGATCTTCG  468 GSNKRPS  619GGCAGTAATAAGCGACCCTCA  469 DNNKRVS  620 GACAATAACAAGCGAGTTTCA  470NNNRRPS  621 AACAATAATAGGCGACCCTCA  471 DNFKRPS  622GACAATTTTAAGCGACCCTCA  472 ENDKRPS  623 GAAAATGATAAACGACCCTCA  473ENNKRLS  624 GAAAATAATAAGCGACTCTCA  474 ADNKRPS  625GCAGATAATAAGCGACCCTCA  475 EDNERPS  626 GAAGATAATGAGCGCCCCTCA  476DTDQRPS  627 GACACTGATCAGCGACCCTCA  477 DNYQRPS  628GACAATTATCAGCGACCCTCA  478 DENKRPS  629 GACGAGAATAAGCGACCCTCA  479DTNKRPS  630 GACACTAATAAGCGACCCTCA  480 DDYRRPS  631GACGATTATCGGCGACCCTCA  481 DNDKRHS  632 GACAACGATAAGCGGCACTCA  482ENDNRPS  633 GAAAATGATAATCGACCCTCA  483 DDNERPS  634GACGATAATGAGCGCCCCTCA  484 DNKKRPS  635 GACAATAAGAAGCGACCCTCA  485DVDKRPS  636 GACGTTGATAAGCGACCCTCA  486 ENKKRPS  637GAAAATAAAAAACGACCCTCT  487 VNDKRPS  638 GTCAATGATAAGCGACCCTCA  488DNDHRPS  639 GACAATGATCACCGACCCTCA  489 DINKRPS  640GACATTAATAAGCGACCCTCA  490 ANNERPS  641 GCCAATAATGAGCGACCCTCA  491DNENRPS  642 GACAATGAAAACCGACCGTCA  492 GDDKRPS  643GGCGATGATAAGCGACCCTCA  493 ANNQRPS  644 GCCAATAATCAGCGACCTTCA  494DDDKRPS  645 GACGATGATAAGCGACCCTCA  495 YNNKRPS  646TACAATAATAAGCGGCCCTCA  496 EDDKRPS  647 GAAGATGATAAGCGACCCTCA  497ENNNRPS  648 GAAAACAATAACCGACCCTCG  498 DNNLRPS  649GACAATAATCTGCGACCCTCA  499 ESNKRPS  650 GAGAGTAACAAGCGACCCTCA  500DTDKRPS  651 GACACTGATAAGCGGCCCTCA  501 DDDQRPS  652GACGATGATCAGCGACCCTCA  502 VNNKRPS  653 GTGAATAATAAGAGACCCTCC  503DDYKRPS  654 GACGATTATAAGCGACCCTCA  504 DNTKRPS  655GACAATACTAAGCGACCCTCA  505 DDTERPS  656 GACGATACTGAGCGACCCTCA  506GNDKRPS  657 GGCAATGATAAGCGACCCTCA  507 DNEKRPS  658GACAATGAAAAGCGACCCTCA  508 DNDDRPS  659 GACAATGATGACCGACCCTCA  509DDNRRPS  660 GACGATAATAGGCGTCCCTCA  510 GNNKRPS  661GGCAATAATAAGCGACCCTCA  511 ANDKRPS  662 GCCAATGATAAGCGACCCTCA  512DNNKRHS  663 GACAATAATAAGCGACACTCA  513 DDNQRPS  664GACGACAATCAGCGACCCTCA  514 GNDRRPS  665 GGCAATGATAGGCGACCCTCA  515DNHNRPS  666 GACAATCATAACCGACCCTCA  516 DNYERPS  667GACAATTATGAGCGACCCTCA  517 ENNKRSS  668 GAAAATAATAAGCGATCCTCA  518DDHKRPS  669 GACGATCATAAGCGGCCCTCA  519 DNNKRRS  670GACAATAATAAACGACGTTCA  520 DNDKRPS  671 GACAATGATAAGCGACCGTCA  521DKNKRPS  672 GACAAGAATAAGCGACCCTCA  522 DNNKRPS  673GACAATAATAAGCGACCCTCA  523 DIDKRPS  674 GACATTGATAAGCGACCCTCA  524DDKKRPS  675 GACGATAAGAAGCGACCCTCA  525 ANNKRPS  676GCCAATAATAAGCGACCCTCA  526 DNDKGPS  677 GACAATGATAAGGGACCCTCA  527EDNRRPS  678 GAAGATAATAGGCGACCCTCA  528 ENNKRPS  679GAGAATAATAAGCGACCCTCA  529 NNNKRPS  680 AACAATAATAAGCGACCCTCA  530DNNERPS  681 GACAATAATGAGCGACCCTCA  531 DNIQRPS  682GACAATATTCAGCGACCCTCA  532 DNNYRPS  683 GACAATAATTACCGACCCTCA  533DNYNRPS  684 GACAATTATAACCGACCCTCA IGLV1-51-L3  685 CGTWDTSLSAVVF 1509TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTGTTC  686 CGTWDTSLSAGVF 1510TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGTGTTC  687 CGTWDTSLSAWVF 1511TGCGGAACATGGGATACCAGCCTGAGTGCTTGGGTGTTC  688 CGTWDRSLSAGVF 1512TGCGGAACATGGGATAGGAGCCTGAGTGCGGGGGTGTTC  689 CGTWDRSLSAWVF 1513TGCGGAACATGGGATAGGAGCCTGAGTGCTTGGGTATTT  690 CGTWDTSLSGGVF 1514TGCGGAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC  691 CGTWDTSLRAGVF 1515TGCGGAACATGGGATACTAGCCTGCGTGCTGGCGTCTTC  692 CGTWDRSLSVWVF 1516TGCGGAACATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  693 CGTWDTSLSVVVF 1517TGCGGAACATGGGATACCAGTCTGAGTGTTGTGGTCTTC  694 CGTWDTSLSAAVF 1518TGCGGAACGTGGGATACCAGCCTGAGTGCTGCGGTGTTC  695 CGAWDTSLSAGVF 1519TGCGGAGCATGGGATACCAGCCTGAGTGCTGGAGTGTTC  696 CATWDTSLSAVVF 1520TGCGCAACATGGGATACCAGCCTGAGTGCTGTGGTATTC  697 CATWDTSLSAGVF 1521TGCGCAACATGGGATACCAGCCTGAGTGCTGGTGTGTTC  698 CGTWESSLSAWVF 1522TGTGGAACATGGGAGAGCAGCCTGAGTGCTTGGGTGTTC  699 CGTWDTTLSAGVF 1523TGCGGAACATGGGATACCACCCTGAGTGCGGGTGTCTTC  700 CGTWDTSLSVWVF 1524TGCGGAACATGGGATACTAGCCTGAGTGTGTGGGTGTTC  701 CGTWDTSLSVGVF 1525TGCGGAACATGGGATACTAGCCTGAGTGTTGGGGTGTTC  702 CGTWDTSLSTGVF 1526TGCGGAACATGGGACACCAGTCTGAGCACTGGCGTCTTC  703 CGTWDTSLSGVVF 1527TGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTCTTC  704 CGTWDTSLSAYVF 1528TGCGGAACATGGGATACCAGCCTGAGTGCTTATGTCTTC  705 CGTWDTSLSAEVF 1529TGCGGAACATGGGATACCAGCCTGAGTGCTGAGGTGTTC  706 CGTWDTGLSAGVF 1530TGCGGAACATGGGATACCGGCCTGAGTGCTGGGGTATTC  707 CGTWDRSLSAYVF 1531TGCGGAACGTGGGATAGGAGCCTGAGTGCTTATGTCTTC  708 CGTWDRSLSAVVF 1532TGCGGAACATGGGATAGGAGCCTCAGTGCCGTGGTATTC  709 CGTWDNTLSAWVF 1533TGCGGAACATGGGATAACACCCTGAGTGCGTGGGTGTTC  710 CGTWDNRLSAGVF 1534TGCGGAACATGGGATAACAGGCTGAGTGCTGGGGTGTTC  711 CGTWDISLSAWVF 1535TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGTGTTC  712 CGTWHSSLSAGVF 1536TGCGGAACATGGCATAGCAGCCTGAGTGCTGGGGTATTC  713 CGTWGSSLSAWVF 1537TGCGGAACATGGGGTAGCAGTTTGAGTGCTTGGGTGTTC  714 CGTWESSLSGWVF 1538TGCGGAACATGGGAGAGCAGCCTGAGTGGTTGGGTGTTC  715 CGTWESSLSAVVF 1539TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTGGTTTTC  716 CGTWDYSLSAVVF 1540TGCGGAACATGGGATTACAGCCTGAGTGCTGTGGTATTC  717 CGTWDYSLSAGVF 1541TGCGGAACATGGGATTACAGCCTGAGTGCTGGGGTATTC  718 CGTWDVSLSVGVF 1542TGCGGAACATGGGATGTCAGCCTGAGTGTTGGAGTGTTC  719 CGTWDTTLSAVVF 1543TGCGGAACATGGGATACCACCCTGAGTGCTGTGGTTTTC  720 CGTWDTTLNIGVF 1544TGCGGAACATGGGATACCACTCTGAATATTGGGGTGTTC  721 CGTWDTSLTAVVF 1545TGCGGAACATGGGATACCAGCCTGACTGCTGTGGTATTC  722 CGTWDTSLTAAVF 1546TGCGGAACCTGGGATACCAGCCTGACTGCTGCTGTGTTC  723 CGTWDTSLSVGLF 1547TGCGGCACATGGGATACCAGCCTGAGTGTGGGGCTATTC  724 CGTWDTSLSGRVF 1548TGCGGAACCTGGGATACCAGCCTGAGTGGTAGGGTGTTC  725 CGTWDTSLSGAVF 1549TGCGGAACATGGGATACCAGCCTGAGTGGTGCAGTGTTC  726 CGTWDTSLSAGLF 1550TGCGGAACATGGGATACCAGCCTGAGTGCTGGCCTGTTC  727 CGTWDTSLSAGGVF 1551TGCGGAACATGGGATACCAGCCTGAGTGCTGGAGGGGTCTTC  728 CGTWDTSLRAYVF 1552TGCGGAACATGGGATACCAGCCTGCGTGCTTATGTCTTC  729 CGTWDTSLRAWVF 1553TGCGGAACATGGGATACTAGTTTGCGTGCTTGGGTATTC  730 CGTWDTSLNTGVF 1554TGCGGAACATGGGATACCAGCCTGAATACTGGGGTATTC  731 CGTWDTSLNIWVF 1555TGCGGAACATGGGATACCAGCCTGAATATTTGGGTGTTC  732 CGTWDTSLNIGVF 1556TGCGGAACATGGGATACAAGCCTGAATATTGGGGTGTTC  733 CGTWDTSLIAVVF 1557TGCGGAACATGGGATACCAGCCTGATTGCTGTGGTGTTC  734 CGTWDRSLSGWVF 1558TGCGGAACGTGGGATAGGAGCCTGAGTGGTTGGGTGTTC  735 CGTWDNRLSGWVF 1559TGCGGAACATGGGATAACAGGCTGAGTGGTTGGGTGTTC  736 CGTWDKSLSAVVF 1560TGCGGAACGTGGGATAAGAGCCTGAGTGCTGTGGTCTTC  737 CGTWDKGLSAWVF 1561TGCGGAACATGGGATAAAGGCCTGAGTGCTTGGGTGTTC  738 CGTWDISLSAGVF 1562TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGTTC  739 CGTWDESLSGGEVVF 1563TGCGGAACATGGGATGAGAGCCTGAGTGGTGGCGAGGTGGTCTTC  740 CGTWDASLSAWVF 1564TGCGGAACATGGGATGCCAGCCTGAGTGCCTGGGTGTTC  741 CGTWDAGLSAWVF 1565TGCGGAACTTGGGATGCCGGCCTGAGTGCTTGGGTGTTC  742 CGAWDTSLSAWVF 1566TGCGGAGCATGGGATACCAGCCTGAGTGCTTGGGTGTTC  743 CGAWDTSLSAVVF 1567TGCGGAGCATGGGATACCAGCCTGAGTGCTGTGGTGTTC  744 CGAWDTSLRAGVF 1568TGCGGAGCATGGGATACCAGCCTGCGTGCTGGGGTTTTC  745 CATWDTSVSAWVF 1569TGCGCAACATGGGATACCAGCGTGAGTGCTTGGGTGTTC  746 CATWDTSLSAWVF 1570TGCGCAACATGGGATACCAGCCTGAGTGCGTGGGTGTTC  747 CATWDNTLSAGVF 1571TGCGCAACATGGGACAACACCCTGAGTGCTGGGGTGTTC  748 CAAWDRSLSVWVF 1572TGCGCAGCATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  749 CYTWHSSLRGGVF 1573TGCTACACATGGCATTCCAGTCTGCGTGGTGGGGTGTTC  750 CVTWTSSPSAWVF 1574TGCGTAACGTGGACTAGTAGCCCGAGTGCTTGGGTGTTC  751 CVTWRGGLVLF 1575TGCGTGACATGGCGTGGTGGCCTTGTGTTGTTC  752 CVTWDTSLTSVVL 1576TGCGTAACATGGGATACCAGCCTGACTTCTGTGGTACTC  753 CVTWDTSLSVYWVF 1577TGCGTAACATGGGATACCAGCCTGAGTGTTTATTGGGTGTTC  754 CVTWDTSLSAWVF 1578TGCGTTACATGGGATACCAGCCTGAGTGCCTGGGTGTTC  755 CVTWDTDLSVALF 1579TGCGTCACATGGGATACCGACCTCAGCGTTGCGCTCTTC  756 CVTWDRSLSGWVF 1580TGCGTAACATGGGATAGGAGCCTGAGTGGTTGGGTGTTC  757 CVTWDRSLREVLF 1581TGCGTAACATGGGATCGCAGCCTGAGAGAGGTGTTATTC  758 CVTWDRSLRAVVF 1582TGCGTAACATGGGATCGCAGCCTGAGAGCGGTGGTATTC  759 CVTWDRSLDAGVF 1583TGCGTAACATGGGACAGGAGCCTCGATGCTGGGGTTTTC  760 CVTWDNTLSAGVF 1584TGCGTGACATGGGATAACACCCTGAGTGCTGGGGTCTTC  761 CVTWDNNLFGVVF 1585TGCGTAACATGGGATAACAACCTGTTTGGTGTGGTCTTC  762 CVSWDTSLSGAVF 1586TGCGTATCATGGGATACCAGCCTGAGTGGTGCGGTATTC  763 CVSWDTSLSAGVF 1587TGCGTCTCATGGGATACCAGCCTGAGTGCTGGGGTATTC  764 CTTWFRTPSDVVF 1588TGCACAACATGGTTTAGGACTCCGAGTGATGTGGTCTTC  765 CTTWFRTASDVVF 1589TGCACAACATGGTTTAGGACTGCGAGTGATGTGGTCTTC  766 CTTWDYGLSVVF 1590TGCACAACGTGGGATTACGGTCTGAGTGTCGTCTTC  767 CTARDTSLSPGGVF 1591TGCACAGCAAGGGATACCAGCCTGAGTCCTGGCGGGGTCTTC  768 CSTWNTRPSDVVF 1592TGCTCAACATGGAATACGAGGCCGAGTGATGTGGTGTTC  769 CSTWESSLTTVVF 1593TGTTCAACATGGGAGAGCAGTTTGACTACTGTGGTCTTC  770 CSTWDTSLTNVLF 1594TGCTCAACATGGGATACCAGCCTCACTAATGTGCTATTC  771 CSTWDTSLSGVVF 1595TGCTCAACATGGGATACCAGCCTGAGTGGAGTAGTCTTC  772 CSTWDHSLKAALF 1596TGCTCAACATGGGATCACAGCCTGAAAGCTGCACTGTTC  773 CSTWDARLSVRVF 1597TGCTCAACCTGGGATGCGAGGCTGAGTGTCCGGGTGTTC  774 CSSYTSSSTWVF 1598TGCTCCTCATATACAAGCAGCAGCACTTGGGTGTTC  775 CSSYATRGLRVLF 1599TGCAGCTCATACGCAACCCGCGGCCTTCGTGTGTTGTTC  776 CSSWDATLSVRIF 1600TGTTCATCATGGGACGCCACCCTGAGTGTTCGCATATTC  777 CQVWEGSSDHWVF 1601TGTCAGGTGTGGGAGGGTAGTAGTGATCATTGGGTGTTC  778 CQTWDNRLSAVVF 1602TGCCAAACCTGGGATAACAGACTGAGTGCTGTGGTGTTC  779 CQTWDHSLHVGVF 1603TGTCAAACGTGGGATCACAGCCTGCATGTTGGGGTGTTC  780 CQSYDDILNVWVL 1604TGCCAGTCCTATGACGACATCTTGAATGTTTGGGTCCTT  781 CNTWDKSLTSELF 1605TGCAATACATGGGATAAGAGTTTGACTTCTGAACTCTTC  782 CLTWDRSLNVRVF 1606TGCTTAACATGGGATCGCAGCCTGAATGTGAGGGTGTTC  783 CLTWDHSLTAYVF 1607TGCCTAACATGGGACCACAGCCTGACTGCTTATGTCTTC  784 CLTRDTSLSAPVF 1608TGCTTAACAAGGGATACCAGTCTGAGTGCCCCTGTGTTC  785 CKTWESGLNFGHVF 1609TGCAAAACATGGGAAAGTGGCCTTAATTTTGGCCACGTCTTC  786 CKTWDTSLSAVVF 1610TGCAAAACATGGGATACCAGCCTGAGTGCTGTGGTCTTC  787 CGVWDVSLGAGVF 1611TGCGGAGTCTGGGATGTCAGTCTGGGTGCTGGGGTGTTC  788 CGVWDTTPSAVLF 1612TGCGGAGTCTGGGATACCACCCCGAGTGCCGTTCTTTTC  789 CGVWDTTLSAVLF 1613TGCGGAGTCTGGGATACCACCCTGAGTGCCGTTCTTTTC  790 CGVWDTSLGVF 1614TGCGGAGTATGGGATACCAGCCTGGGGGTCTTC  791 CGVWDTNLGKWVF 1615TGCGGGGTATGGGATACCAACCTGGGTAAATGGGTTTTC  792 CGVWDTGLDAGWVF 1616TGTGGAGTTTGGGATACTGGCCTGGATGCTGGTTGGGTGTTC  793 CGVWDNVLEAYVF 1617TGCGGAGTGTGGGATAACGTCCTGGAGGCCTATGTCTTC  794 CGVWDISLSANWVF 1618TGCGGAGTCTGGGATATCAGCCTGAGTGCTAATTGGGTGTTC  795 CGVWDHSLGIWAF 1619TGCGGAGTATGGGATCACAGCCTGGGGATTTGGGCCTTC  796 CGVWDDILTAEVF 1620TGCGGAGTTTGGGATGATATTCTGACTGCTGAAGTGTTC  797 CGVRDTSLGVF 1621TGCGGAGTTCGGGATACCAGCCTGGGGGTCTTC  798 CGTYDTSLPAWVF 1622TGCGGAACATACGATACGAGCCTGCCTGCTTGGGTGTTT  799 CGTYDNLVFGYVF 1623TGCGGAACTTACGATAATCTTGTATTTGGTTATGTCTTC  800 CGTYDDRLREVF 1624TGCGGAACATACGATGATAGACTCAGAGAGGTGTTC  801 CGTWVTSLSAGVF 1625TGCGGAACGTGGGTTACCAGCCTGAGTGCTGGGGTGTTC  802 CGTWVSSLTTVVF 1626TGCGGAACATGGGTTAGCAGCCTGACTACTGTAGTATTC  803 CGTWVSSLNVWVF 1627TGCGGAACATGGGTTAGCAGCCTGAACGTCTGGGTGTTC  804 CGTWVGRFWVF 1628TGCGGAACATGGGTTGGCAGGTTTTGGGTATTC  805 CGTWSGGPSGHWLF 1629TGCGGAACATGGTCTGGCGGCCCGAGTGGCCATTGGTTGTTC  806 CGTWSGGLSGHWLF 1630TGCGGAACATGGTCTGGCGGCCTGAGTGGCCATTGGTTGTTC  807 CGTWQTGREAVLF 1631TGCGGAACGTGGCAGACCGGCCGGGAGGCTGTCCTATTT  808 CGTWQSRLRWVF 1632TGCGGAACGTGGCAGAGCAGGCTGAGGTGGGTGTTC  809 CGTWQSRLGWVF 1633TGCGGAACGTGGCAGAGCAGGCTGGGGTGGGTGTTC  810 CGTWPRSLSAVWVF 1634TGCGGAACATGGCCTAGGAGCCTGAGTGCTGTTTGGGTGTTC  811 CGTWNNYLSAGDVVF 1635TGCGGAACATGGAATAACTACCTGAGTGCTGGCGATGTGGTTTTC  812 CGTWLGSQSPYWVF 1636TGCGGAACATGGCTTGGCAGCCAGAGTCCTTATTGGGTCTTC  813 CGTWHTGLSAYVF 1637TGCGGAACATGGCATACCGGCCTGAGTGCTTATGTCTTC  814 CGTWHSTLSAGHWVF 1638TGCGGAACATGGCATAGTACCCTGAGTGCTGGCCATTGGGTGTTC  815 CGTWHSSLSTWVF 1639TGCGGAACATGGCATAGTAGCCTGAGTACTTGGGTGTTC  816 CGTWHSSLSAYVF 1640TGCGGAACATGGCATAGCAGCCTGAGTGCCTATGTCTTC  817 CGTWHSSLSAVVF 1641TGCGGAACATGGCATAGCAGCCTGAGTGCTGTGGTATTC  818 CGTWHSGLSGWVF 1642TGCGGAACGTGGCATTCCGGCCTGAGTGGGTGGGTTTTC  819 CGTWHNTLRNVIF 1643TGCGGAACATGGCATAACACCCTGCGTAATGTGATATTC  820 CGTWHASLTAVF 1644TGCGGAACATGGCATGCCAGCCTGACTGCTGTGTTC  821 CGTWGWYGSQRGVVF 1645TGCGGGACATGGGGATGGTATGGCAGCCAGAGAGGCGTCGTCTTC  822 CGTWGWYGGQRGVVF 1646TGCGGGACATGGGGATGGTATGGCGGCCAGAGAGGCGTCGTCTTC  823 CGTWGTSLSAWVF 1647TGCGGAACCTGGGGAACCAGCCTGAGTGCTTGGGTGTTC  824 CGTWGSSLTTGLF 1648TGCGGAACCTGGGGTAGCAGCCTGACTACTGGCCTGTTC  825 CGTWGSSLTAYVF 1649TGCGGAACATGGGGTAGCAGCCTGACTGCCTATGTCTTC  826 CGTWGSSLSVVF 1650TGCGGAACATGGGGTAGCAGCCTGAGTGTTGTGTTC  827 CGTWGSSLSGGVF 1651TGCGGAACATGGGGTAGCAGCCTGAGTGGTGGGGTGTTC  828 CGTWGSSLSAYWVF 1652TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATTGGGTGTTC  829 CGTWGSSLSAYVVF 1653TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTGGTGTTC  830 CGTWGSSLSAYVF 1654TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTCTTC  831 CGTWGSSLSAVVF 1655TGCGGAACGTGGGGTAGTAGCCTGAGTGCTGTGGTGTTC  832 CGTWGSSLSAPYVF 1656TGCGGAACATGGGGTAGCAGCCTGAGTGCTCCTTATGTCTTC  833 CGTWGSSLSAPVF 1657TGCGGAACATGGGGTAGCAGCCTGAGTGCCCCGGTGTTC  834 CGTWGSSLSAGVF 1658TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGTGTTC  835 CGTWGSSLSAGLF 1659TGCGGAACTTGGGGTAGCAGCCTGAGTGCTGGACTGTTC  836 CGTWGSSLSAGALF 1660TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGCACTCTTC  837 CGTWGSSLRAWVF 1661TGCGGAACATGGGGCAGTAGCCTGCGTGCTTGGGTGTTC  838 CGTWFTSLASGVF 1662TGCGGAACCTGGTTTACTAGTCTGGCTAGTGGGGTTTTC  839 CGTWETSLSVVVI 1663TGCGGAACTTGGGAGACCAGTCTGAGTGTCGTGGTCATC  840 CGTWETSLSGVF 1664TGCGGAACATGGGAGACCAGCCTGAGTGGTGTCTTC  841 CGTWETSLSDWVF 1665TGCGGAACATGGGAAACCAGCCTGAGTGATTGGGTATTC  842 CGTWETSLSAGVF 1666TGCGGAACATGGGAGACCAGCCTGAGTGCTGGGGTATTC  843 CGTWETSLNYVAF 1667TGCGGAACATGGGAAACCAGCCTTAATTATGTGGCCTTC  844 CGTWETSLNTWLL 1668TGCGGAACATGGGAGACCAGCCTGAATACTTGGTTGCTC  845 CGTWETSESGNYIF 1669TGCGGAACATGGGAGACCAGCGAGAGTGGTAATTACATCTTC  846 CGTWETRLGTWVI 1670TGCGGAACATGGGAAACCAGACTGGGTACTTGGGTGATC  847 CGTWETQLYWVF 1671TGCGGAACATGGGAGACCCAGTTATATTGGGTGTTC  848 CGTWETGLSAGEVF 1672TGCGGAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC  849 CGTWESTLSVFLF 1673TGCGGAACTTGGGAAAGCACCCTGAGTGTTTTCCTATTC  850 CGTWESSLTVVVF 1674TGCGGGACATGGGAAAGTAGCCTGACTGTTGTGGTCTTC  851 CGTWESSLTGVVF 1675TGCGGAACATGGGAAAGTAGCCTGACTGGAGTGGTATTC  852 CGTWESSLTGFVF 1676TGCGGAACATGGGAAAGCAGCCTGACTGGTTTTGTCTTC  853 CGTWESSLSVGVF 1677TGTGGAACATGGGAGAGCAGCCTGAGTGTTGGGGTGTTC  854 CGTWESSLSEWVF 1678TGCGGAACCTGGGAAAGTAGCCTCAGTGAATGGGTGTTC  855 CGTWESSLSAVF 1679TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTATTC  856 CGTWESSLSAGYIF 1680TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGTTATATCTTC  857 CGTWESSLSAGVF 1681TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGAGTGTTC  858 CGTWESSLSAGPVF 1682TGCGGAACATGGGAAAGCAGCCTGAGCGCTGGCCCGGTGTTC  859 CGTWESSLSAGGQVF 1683TGCGGAACATGGGAAAGCAGCCTGAGTGCTGGAGGCCAGGTGTTC  860 CGTWESSLSAFGGYVF 1684TGCGGAACATGGGAGAGCAGCCTGAGTGCCTTCGGCGGTTATGTCTTC  861 CGTWESSLRVWVF 1685TGCGGAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC  862 CGTWESSLFTGPWVF 1686TGCGGAACATGGGAAAGCAGCCTCTTTACTGGGCCTTGGGTGTTC  863 CGTWESLSATYVF 1687TGCGGAACATGGGAGAGCCTGAGTGCCACCTATGTCTTC  864 CGTWESGLSAGVF 1688TGCGGAACATGGGAGAGCGGCCTGAGTGCTGGTGTCTTC  865 CGTWESDFWVF 1689TGCGGAACATGGGAAAGCGACTTTTGGGTGTTT  866 CGTWENRLSAVVF 1690TGCGGTACATGGGAAAACAGACTGAGTGCTGTGGTCTTC  867 CGTWENRLSAGVF 1691TGCGGAACATGGGAAAACAGACTGAGTGCCGGGGTATTC  868 CGTWEISLTTSVVF 1692TGCGGAACATGGGAAATCAGCCTGACTACTTCTGTGGTATTC  869 CGTWEISLSTSVVF 1693TGCGGAACATGGGAAATCAGCCTGAGTACTTCTGTGGTATTC  870 CGTWEGSLSVVF 1694TGCGGAACATGGGAAGGCAGCCTCAGTGTTGTTTTC  871 CGTWEGSLRVF 1695TGCGGAACATGGGAAGGCAGCCTGAGGGTGTTC  872 CGTWEGSLRHVF 1696TGCGGAACATGGGAGGGCAGCCTGAGGCACGTGTTC  873 CGTWDYSPVRAGVF 1697TGCGGAACATGGGATTACAGCCCTGTACGTGCTGGGGTGTTC  874 CGTWDYSLSVYLF 1698TGCGGAACGTGGGATTACAGCCTGAGTGTTTATCTCTTC  875 CGTWDYSLSSGVVF 1699TGCGGAACATGGGATTACAGCCTGAGTTCTGGCGTGGTATTC  876 CGTWDYSLSAWVF 1700TGCGGAACATGGGATTACAGCCTGAGTGCCTGGGTGTTC  877 CGTWDYSLSAEVF 1701TGCGGAACATGGGATTACAGTCTGAGTGCTGAGGTGTTC  878 CGTWDYSLRRAIF 1702TGCGGAACATGGGATTACAGCCTGCGTCGTGCGATATTC  879 CGTWDWSLILQLF 1703TGCGGAACATGGGATTGGAGCCTCATTCTTCAATTGTTC  880 CGTWDVTLHTGVF 1704TGCGGAACATGGGATGTCACCTTGCATACTGGGGTGTTC  881 CGTWDVTLHIGVF 1705TGCGGAACATGGGATGTCACCTTGCATATTGGGGTGTTC  882 CGTWDVTLHAGVF 1706TGCGGAACATGGGATGTCACCTTGCATGCTGGGGTGTTC  883 CGTWDVSLYSGGVF 1707TGCGGAACATGGGATGTCAGTTTGTATAGTGGCGGGGTCTTC  884 CGTWDVSLTSFVF 1708TGTGGAACATGGGATGTCAGCCTGACTTCTTTCGTCTTC  885 CGTWDVSLSVGVL 1709TGCGGAACATGGGATGTCAGCCTGAGTGTTGGGGTGCTC  886 CGTWDVSLSAGDVVF 1710TGCGGAACGTGGGATGTCAGCCTGAGTGCTGGCGATGTAGTTTTC  887 CGTWDVSLNVVVF 1711TGCGGAACATGGGATGTCAGCCTGAATGTCGTGGTTTTC  888 CGTWDVSLNTQVF 1712TGCGGAACATGGGATGTCAGCCTGAATACTCAGGTGTTC  889 CGTWDVSLGALF 1713TGCGGCACATGGGATGTGAGCCTGGGTGCGCTGTTC  890 CGTWDVNLKTVVF 1714TGCGGAACGTGGGACGTTAATCTGAAAACTGTCGTTTTC  891 CGTWDVILSAEVF 1715TGCGGAACATGGGATGTCATCCTGAGTGCTGAGGTATTC  892 CGTWDTTVSAVVF 1716TGCGGAACATGGGATACCACCGTGAGTGCTGTGGTTTTC  893 CGTWDTTLTAWVF 1717TGCGGAACATGGGATACCACCCTGACTGCCTGGGTGTTC  894 CGTWDTTLSVFLF 1718TGCGGAACATGGGACACCACCTTGAGTGTTTTCCTATTC  895 CGTWDTSVSAGVF 1719TGCGGGACTTGGGATACCAGTGTGAGTGCTGGGGTGTTC  896 CGTWDTSVISWVF 1720TGCGGAACATGGGATACCAGTGTGATTTCTTGGGTTTTC  897 CGTWDTSRSSLYVVF 1721TGCGGAACATGGGATACCAGTCGGAGTTCTCTCTATGTGGTCTTC  898 CGTWDTSRSAWVF 1722TGCGGAACATGGGATACCAGCCGGAGTGCTTGGGTATTC  899 CGTWDTSRNPGGIF 1723TGCGGAACATGGGATACCAGCCGGAATCCTGGAGGAATTTTC  900 CGTWDTSRGHVF 1724TGCGGAACATGGGACACCAGTCGGGGTCATGTTTTC  901 CGTWDTSPSTGQVLF 1725TGCGGAACATGGGATACCAGCCCGAGTACTGGCCAGGTGCTTTTC  902 CGTWDTSPSAWVF 1726TGCGGAACATGGGATACCAGCCCGAGTGCCTGGGTGTTC  903 CGTWDTSLTWVF 1727TGCGGAACATGGGATACTAGCCTGACCTGGGTGTTC  904 CGTWDTSLTWFAVF 1728TGCGGAACATGGGATACCAGCCTGACGTGGTTCGCAGTGTTC  905 CGTWDTSLTVVVF 1729TGCGGAACATGGGATACCAGCCTGACTGTTGTGGTATTC  906 CGTWDTSLTTSWVF 1730TGCGGAACATGGGATACCAGCCTGACTACTTCTTGGGTGTTC  907 CGTWDTSLTTGPFWCF 1731TGCGGAACATGGGATACCAGCCTGACCACTGGTCCTTTTTGGGTGTTC  908 CGTWDTSLTPFYVF1732 TGCGGAACATGGGATACCAGCCTGACTCCTTTTTATGTCTTC  909 CGTWDTSLTAYVF 1733TGCGGAACATGGGATACCAGCCTGACTGCTTATGTCTTC  910 CGTWDTSLTAWVF 1734TGCGGAACATGGGATACCAGCCTGACTGCTTGGGTGTTC  911 CGTWDTSLTAWGVF 1735TGCGGAACATGGGATACCAGCCTGACTGCGTGGGGGGTGTTC  912 CGTWDTSLTAVVL 1736TGCGGCACATGGGATACCAGCCTGACTGCGGTGGTTCTC  913 CGTWDTSLTARVF 1737TGCGGAACCTGGGATACCAGCCTGACTGCTCGGGTTTTC  914 CGTWDTSLTAIVF 1738TGCGGAACATGGGATACCAGCCTGACTGCGATTGTCTTC  915 CGTWDTSLTAGVF 1739TGCGGAACATGGGATACCAGCCTGACTGCTGGTGTCTTC  916 CGTWDTSLSVYVF 1740TGCGGAACATGGGATACCAGCCTGAGTGTTTATGTCTTC  917 CGTWDTSLSVVF 1741TGCGGAACATGGGATACCAGCCTGAGTGTGGTGTTC  918 CGTWDTSLSVGEF 1742TGCGGGACATGGGATACCAGCCTGAGTGTTGGGGAATTC  919 CGTWDTSLSTWVF 1743TGCGGAACATGGGATACCAGCCTGAGTACTTGGGTGTTC  920 CGTWDTSLSTVVF 1744TGCGGAACATGGGATACCAGCCTGAGTACTGTGGTATTC  921 CGTWDTSLSTGQVLF 1745TGCGGAACATGGGATACCAGCCTGAGTACTGGCCAGGTGCTTTTC  922 CGTWDTSLSTGPLWVF 1746TGCGGCACATGGGATACCAGCCTGAGCACTGGTCCTCTTTGGGTGTTC  923 CGTWDTSLSSYVF 1747TGCGGAACTTGGGATACCAGCCTGAGTTCTTATGTCTTC  924 CGTWDTSLSSVVF 1748TGCGGAACATGGGATACCAGCCTGAGTTCTGTGGTCTTC  925 CGTWDTSLSSRYIF 1749TGCGGAACATGGGATACCAGCCTGAGTTCTAGATACATATTC  926 CGTWDTSLSSRFIF 1750TGCGGAACATGGGATACCAGCCTGAGTTCTAGATTCATATTC  927 CGTWDTSLSSGWVF 1751TGCGGAACATGGGATACCAGCCTGAGTTCTGGGTGGGTGTTC  928 CGTWDTSLSRYVF 1752TGCGGAACATGGGATACCAGCCTGAGTCGGTATGTGTTC  929 CGTWDTSLSQWLF 1753TGCGGAACTTGGGATACCAGTCTGAGTCAATGGCTGTTC  930 CGTWDTSLSPGLWVF 1754TGCGGAACATGGGATACCAGCCTGAGTCCTGGCCTTTGGGTGTTC  931 CGTWDTSLSNYVF 1755TGCGGAACATGGGATACCAGCCTGAGTAATTATGTCTTC  932 CGTWDTSLSIWVF 1756TGCGGAACATGGGATACCAGCCTAAGTATTTGGGTGTTC  933 CGTWDTSLSIGPFWVF 1757TGCGGCACATGGGATACCAGCCTGAGCATTGGTCCTTTTTGGGTGTTC  934 CGTWDTSLSGWVF 1758TGCGGAACATGGGATACCAGCCTGAGTGGTTGGGTGTTC  935 CGTWDTSLSGTVF 1759TGCGGAACATGGGATACCAGCCTGAGTGGTACAGTGTTC  936 CGTWDTSLSGGQVF 1760TGCGGAACATGGGATACTAGTCTGAGTGGTGGCCAGGTGTTC  937 CGTWDTSLSGGIF 1761TGCGGAACATGGGATACCAGCCTGAGTGGTGGGATATTC  938 CGTWDTSLSGEDVVI 1762TGCGGAACATGGGATACCAGCCTGAGTGGTGAGGATGTGGTAATC  939 CGTWDTSLSFLYAF 1763TGCGGAACATGGGATACCAGCCTGAGTTTCCTTTATGCTTTC  940 CGTWDTSLSEVVF 1764TGCGGAACATGGGATACCAGCCTGAGTGAGGTCGTATTC  941 CGTWDTSLSEVF 1765TGCGGAACATGGGATACCAGCCTGAGTGAAGTGTTC  942 CGTWDTSLSENWVF 1766TGCGGAACATGGGATACTAGCCTGAGTGAAAATTGGGTGTTC  943 CGTWDTSLSAYIF 1767TGCGGAACATGGGATACCAGCCTGAGTGCCTACATATTC  944 CGTWDTSLSAVVL 1768TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTACTC  945 CGTWDTSLSAVF 1769TGCGGAACATGGGATACCAGCCTGAGTGCTGTTTTC  946 CGTWDTSLSARVF 1770TGCGGAACATGGGATACCAGCCTGAGTGCCCGGGTGTTC  947 CGTWDTSLSARQVF 1771TGCGGCACATGGGATACCAGCCTGAGTGCCCGCCAGGTATTC  948 CGTWDTSLSALVF 1772TGCGGAACATGGGATACCAGCCTGAGTGCTTTGGTTTTC  949 CGTWDTSLSAKVF 1773TGCGGAACATGGGATACCAGCCTGAGTGCTAAGGTGTTC  950 CGTWDTSLSAKIF 1774TGCGGAACATGGGATACCAGCCTGAGTGCGAAAATCTTC  951 CGTWDTSLSAKAVF 1775TGCGGAACATGGGATACCAGCCTGAGTGCCAAGGCGGTATTC  952 CGTWDTSLSAHAVF 1776TGCGGAACATGGGATACCAGCCTGAGTGCCCATGCTGTGTTC  953 CGTWDTSLSAGYVF 1777TGCGGAACATGGGATACCAGCCTGAGTGCTGGCTATGTCTTC  954 CGTWDTSLSAGRWVF 1778TGCGGAACATGGGACACCAGTCTGAGTGCTGGCCGCTGGGTGTTC  955 CGTWDTSLSAGIF 1779TGCGGAACATGGGATACCAGCCTGAGTGCTGGGATATTC  956 CGTWDTSLSAGGFRVF 1780TGCGGAACATGGGATACCAGCCTGAGTGCTGGTGGGTTCCGGGTCTTC  957 CGTWDTSLSAGAF 1781TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGCATTC  958 CGTWDTSLSADWN, 1782TGCGGAACATGGGATACCAGTCTGAGTGCTGATTGGTTTTTC  959 CGTWDTSLSADEYVF 1783TGCGGAACATGGGATACCAGCCTGAGTGCTGATGAATATGTCTTC  960 CGTWDTSLSAAWVF 1784TGCGGCACATGGGATACCAGCCTGAGTGCGGCTTGGGTGTTC  961 CGTWDTSLSAALF 1785TGCGGAACATGGGATACCAGCCTGAGTGCTGCGCTATTC  962 CGTWDTSLSAAGVF 1786TGCGGAACATGGGATACCAGCCTGAGTGCTGCGGGGGTTTTC  963 CGTWDTSLRVVVF 1787TGCGGAACATGGGATACCAGCCTGAGAGTTGTGGTTTTC  964 CGTWDTSLRTWVF 1788TGCGGAACATGGGATACCAGCCTGAGAACCTGGGTATTC  965 CGTWDTSLRGAVF 1789TGCGGAACGTGGGATACCAGCCTGAGGGGTGCAGTGTTC  966 CGTWDTSLRAVVF 1790TGCGGAACATGGGATACCAGCCTGCGTGCTGTGGTATTC  967 CGTWDTSLNVVYVF 1791TGCGGAACATGGGATACAAGCCTGAATGTAGTTTATGTCTTC  968 CGTWDTSLNTYLF 1792TGCGGAACATGGGATACCAGCCTCAACACCTACCTGTTC  969 CGTWDTSLNFAWLF 1793TGCGGAACATGGGATACTAGCCTGAACTTCGCTTGGCTGTTC  970 CGTWDTSLLVWLF 1794TGCGGCACATGGGATACCAGCCTTCTTGTGTGGCTTTTC  971 CGTWDTSLKTWVF 1795TGCGGAACATGGGATACCAGTCTGAAGACGTGGGTGTTC  972 CGTWDTSLIVWVF 1796TGCGGAACATGGGATACCAGTCTGATTGTCTGGGTGTTC  973 CGTWDTSLITGVF 1797TGCGGAACATGGGATACCAGCCTAATTACTGGGGTGTTC  974 CGTWDTSLISVVF 1798TGCGGAACATGGGATACCAGCCTGATTAGCGTGGTATTC  975 CGTWDTSLIAYVF 1799TGCGGAACATGGGATACCAGCCTGATTGCTTATGTCTTC  976 CGTWDTSLHIELF 1800TGCGGAACATGGGATACCAGCCTGCACACTGAGTTGTTC  977 CGTWDTSLGSYVF 1801TGCGGAACTTGGGATACCAGCCTGGGTTCTTATGTCTTC  978 CGTWDTSLGSLWVF 1802TGCGGAACATGGGATACCAGCCTGGGTTCTCTTTGGGTGTTC  979 CGTWDTSLGSGVF 1803TGCGGTACATGGGATACCAGCCTGGGTTCTGGGGTATTC  980 CGTWDTSLGGRGVF 1804TGCGGAACTTGGGATACCAGTCTGGGTGGTAGAGGGGTCTTC  981 CGTWDTSLGAWVF 1805TGCGGAACATGGGATACCAGCCTGGGTGCTTGGGTGTTC  982 CGTWDTSLGAVVF 1806TGCGGAACATGGGATACCAGCCTGGGTGCCGTGGTATTC  983 CGTWDTSLGAGVF 1807TGCGGAACATGGGATACCAGCCTGGGTGCTGGGGTATTC  984 CGTWDTSLGAGLF 1808TGCGGAACATGGGATACCAGCCTGGGTGCTGGCCTATTC  985 CGTWDTSLDAVVF 1809TGCGGAACATGGGATACCAGTCTGGATGCTGTGGTTTTC  986 CGTWDTSLDAVLF 1810TGCGGGACTTGGGATACCAGCCTGGATGCTGTGCTGTTC  987 CGTWDTSLAWVF 1811TGCGGAACATGGGATACCAGCCTGGCTTGGGTGTTC  988 CGTWDTSLATGLF 1812TGCGGAACATGGGATACCAGCCTGGCGACTGGACTGTTC  989 CGTWDTSLAPVVF 1813TGCGGGACATGGGATACCAGCCTGGCCCCTGTAGTCTTC  990 CGTWDTRLTIVIF 1814TGCGGAACATGGGACACCCGCCTGACTATTGTGATCTTC  991 CGTWDTRLSVWLF 1815TGTGGAACATGGGACACCAGGCTGAGTGTTTGGCTGTTC  992 CGTWDTRLSVGVF 1816TGCGGAACGTGGGACACCAGACTGAGTGTTGGGGTTTTC  993 CGTWDTRLSTVIF 1817TGCGGCACATGGGATACCAGACTGAGTACTGTAATTTTC  994 CGTWDTRLSSVVF 1818TGCGGAACATGGGATACCCGCCTGAGTTCTGTGGTCTTC  995 CGTWDTRLSIVVF 1819TGCGGAACATGGGATACCCGCCTGAGTATTGTGGTTTTC  996 CGTWDTRLSAYVVF 1820TGCGGAACATGGGATACCAGACTGAGTGCCTATGTGGTATTC  997 CGTWDTRLSAWVF 1821TGCGGAACCTGGGACACCCGCCTGAGTGCGTGGGTGTTC  998 CGTWDTRLSAVVF 1822TGCGGAACATGGGATACCAGACTGAGTGCTGTGGTGTTC  999 CGTWDTRLSAGLF 1823TGCGGAACATGGGATACCCGCCTGAGTGCTGGGTTGTTC 1000 CGTWDTRLSAGGVF 1824TGCGGAACATGGGATACCAGACTGAGTGCTGGTGGGGTGTTC 1001 CGTWDTRLNVWLF 1825TGCGGAACATGGGATACCAGATTGAATGTGTGGCTATTC 1002 CGTWDTNREVVLL 1826TGCGGAACATGGGATACCAACCGGGAAGTTGTGCTCCTC 1003 CGTWDTNLRAHVF 1827TGCGGAACATGGGATACCAACCTGCGTGCCCATGTCTTC 1004 CGTWDTNLPAVVF 1828TGCGGAACATGGGATACTAATCTGCCCGCTGTAGTGTTC 1005 CGTWDTNLGGVF 1829TGCGGAACATGGGACACCAATTTGGGTGGGGTGTTC 1006 CGTWDTIVSIGVF 1830TGCGGAACATGGGATACCATCGTGAGTATTGGGGTGTTC 1007 CGTWDTILSAVVF 1831TGCGGAACATGGGATACCATCCTGAGTGCGGTGGTGTTC 1008 CGTWDTILSAEVF 1832TGCGGCACATGGGATACCATCCTGAGTGCTGAGGTGTTC 1009 CGTWDTHLGVVF 1833TGCGGAACATGGGATACCCACCTGGGTGTGGTTTTC 1010 CGTWDTGPSPHWLF 1834TGCGGAACATGGGATACCGGCCCGAGCCCTCATTGGCTGTTC 1011 CGTWDTGLTFGGVF 1835TGCGGAACATGGGATACCGGCCTGACTTTTGGAGGCGTGTTC 1012 CGTWDTGLTAFVF 1836TGCGGAACATGGGATACCGGCCTGACTGCTTTTGTCTTC 1013 CGTWDTGLSVWVF 1837TGCGGAACATGGGATACCGGCCTGAGTGTTTGGGTGTTC 1014 CGTWDTGLSTGIF 1838TGCGGAACATGGGATACCGGCCTGAGTACTGGGATTTTC 1015 CGTWDTGLSSLLF 1839TGCGGAACATGGGATACCGGCCTGAGTTCCCTGCTCTTC 1016 CGTWDTGLSIVVF 1840TGCGGAACGTGGGACACCGGCCTGAGTATTGTGGTGTTC 1017 CGTWDTGLSFVVF 1841TGCGGAACGTGGGACACCGGCCTGAGTTTTGTGGTGTTC 1018 CGTWDTGLSAWVF 1842TGCGGAACATGGGATACCGGCCTGAGTGCTTGGGTGTTC 1019 CGTWDTGLSAGVVF 1843TGCGGAACATGGGATACCGGCCTGAGTGCTGGTGTGGTATTC 1020 CGTWDTGLRGWIF 1844TGCGGAACATGGGATACCGGTCTGAGGGGTTGGATTTTC 1021 CGTWDTELSAGVF 1845TGCGGAACATGGGATACCGAGCTAAGTGCGGGGGTCTTC 1022 CGTWDTALTAGVF 1846TGCGGAACGTGGGATACCGCCCTGACTGCTGGGGTGTTC 1023 CGTWDTALSLVVF 1847TGCGGAACATGGGATACTGCCCTGAGTCTTGTGGTCTTC 1024 CGTWDTALSAWLF 1848TGCGGAACATGGGATACCGCCCTGAGTGCCTGGCTGTTC 1025 CGTWDTALSAGVF 1849TGCGGCACATGGGATACCGCCCTGAGTGCTGGGGTGTTC 1026 CGTWDTALRGVLF 1850TGCGGAACATGGGATACCGCCCTGCGTGGCGTGCTGTTC 1027 CGTWDTALKEWLF 1851TGCGGAACATGGGATACCGCCCTGAAAGAATGGCTGTTC 1028 CGTWDRTLTAGDVLF 1852TGCGGAACATGGGATAGGACCCTGACTGCTGGCGATGTGCTCTTC 1029 CGTWDRSVTYVF 1853TGCGGAACATGGGATAGAAGCGTGACTTATGTCTTC 1030 CGTWDRSRNEWVF 1854TGCGGAACATGGGATCGCAGCCGAAATGAATGGGTGTTC 1031 CGTWDRSLTVWVF 1855TGCGGAACATGGGATCGCAGTCTGACTGTTTGGGTCTTC 1032 CGTWDRSLTPGWLF 1856TGCGGAACATGGGATCGCAGCCTGACTCCTGGGTGGTTGTTC 1033 CGTWDRSLTAWVF 1857TGCGGAACATGGGATAGAAGCCTGACTGCTTGGGTGTTC 1034 CGTWDRSLSVVVF 1858TGCGGAACATGGGACCGCAGCCTGAGTGTTGTGGTATTC 1035 CGTWDRSLSVVF 1859TGCGGCACATGGGATCGCAGCCTGAGTGTAGTCTTC 1036 CGTWDRSLSVQLF 1860TGCGGAACATGGGATAGGAGCCTGAGTGTTCAATTGTTC 1037 CGTWDRSLSVLWVF 1861TGCGGAACATGGGATCGCAGCCTCAGTGTTCTTTGGGTGTTC 1038 CGTWDRSLSVGLF 1862TGCGGAACATGGGATCGCAGCCTGAGTGTTGGATTATTC 1039 CGTWDRSLSTWVF 1863TGCGGAACATGGGATCGCAGCCTGAGTACTTGGGTGTTC 1040 CGTWDRSLSTHWVL 1864TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGCTC 1041 CGTWDRSLSTHWVF 1865TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGTTC 1042 CGTWDRSLSSAVF 1866TGCGGAACCTGGGATCGAAGCCTGAGTTCTGCGGTGTTC 1043 CGTWDRSLSPSYVF 1867TGCGGAACATGGGACAGAAGCCTGAGTCCCTCTTATGTCTTC 1044 CGTWDRSLSGEVF 1868TGCGGAACATGGGATAGGAGCCTGAGTGGTGAGGTGTTC 1045 CGTWDRSLSGAVF 1869TGCGGAACATGGGATAGGAGCCTGAGTGGTGCGGTGTTC 1046 CGTWDRSLSAVAF 1870TGCGGAACATGGGATCGCAGCCTGAGTGCTGTGGCATTC 1047 CGTWDRSLSAGGEF 1871TGCGGAACATGGGATAGGAGCCTGAGTGCCGGGGGGGAATTC 1048 CGTWDRSLSAFWVF 1872TGCGGAACATGGGATCGCAGCCTGAGTGCTTTTTGGGTGTTC 1049 CGTWDRSLSAAVF 1873TGCGGAACATGGGATAGGAGCCTGAGTGCTGCGGTGTTC 1050 CGTWDRSLSAALF 1874TGCGGAACATGGGATAGGAGCCTGAGTGCTGCACTCTTC 1051 CGTWDRSLRVF 1875TGCGGAACATGGGATCGCAGCCTGAGAGTGTTC 1052 CGTWDRSLNWVF 1876TGCGGTACATGGGACAGAAGCCTTAATTGGGTGTTC 1053 CGTWDRSLNVYVF 1877TGCGGAACATGGGATCGCAGCCTGAATGTTTATGTCTTC 1054 CGTWDRSLNVGVF 1878TGCGGAACATGGGATAGGAGCCTGAATGTTGGGGTGTTC 1055 CGTWDRSLHVVF 1879TGCGGAACATGGGATCGGAGCCTGCATGTGGTCTTC 1056 CGTWDRSLGGWVF 1880TGTGGAACATGGGATCGCAGCCTGGGTGGTTGGGTGTTC 1057 CGTWDRSLGAFWVF 1881TGCGGAACATGGGATCGCAGCCTGGGTGCTTTTTGGGTGTTC 1058 CGTWDRSLFWVF 1882TGCGGAACATGGGATAGAAGCCTGTTTTGGGTGTTC 1059 CGTWDRSLAAGVF 1883TGCGGAACGTGGGATCGCAGCCTGGCTGCTGGGGTGTTC 1060 CGTWDRRLSGVVF 1884TGCGGAACATGGGATAGGAGGTTGAGTGGTGTCGTATTC 1061 CGTWDRRLSDVVF 1885TGCGGAACGTGGGATCGCCGCCTAAGTGATGTGGTATTC 1062 CGTWDRRLSAVVF 1886TGCGGAACATGGGATAGGAGGCTGAGTGCTGTGGTATTC 1063 CGTWDRRLNVAFF 1887TGCGGAACATGGGATAGACGCCTGAATGTTGCGTTCTTC 1064 CGTWDRRLLAVF 1888TGTGGAACATGGGATAGGAGGCTGCTTGCTGTTTTC 1065 CGTWDRNLRAVVF 1889TGCGGAACTTGGGATAGGAACCTGCGCGCCGTGGTCTTC 1066 CGTWDRLSAGVF 1890TGCGGAACATGGGATAGGCTGAGTGCTGGGGTGTTC 1067 CGTWDRGPNTGVF 1891TGCGGAACATGGGATAGAGGCCCGAATACTGGGGTATTC 1068 CGTWDRGLNTVYVF 1892TGCGGAACATGGGATAGAGGCCTGAATACTGTTTACGTCTTC 1069 CGTWDNYVSAPWVF 1893TGCGGAACATGGGATAACTATGTGAGTGCCCCTTGGGTGTTC 1070 CGTWDNYLSAGDVVF 1894TGCGGAACATGGGATAACTACCTGAGTGCTGGCGATGTGGTTTTC 1071 CGTWDNYLRAGVF 1895TGCGGAACATGGGATAACTACCTGAGAGCTGGGGTCTTC 1072 CGTWDNYLGAVVF 1896TGCGGAACATGGGACAATTATCTGGGTGCCGTGGTTTTC 1073 CGTWDNYLGAGVF 1897TGCGGAACATGGGATAACTACCTGGGTGCGGGGGTGTTC 1074 CGTWDNTVSAPWVF 1898TGCGGAACATGGGATAACACCGTGAGTGCCCCTTGGGTTTTC 1075 CGTWDNTLSLWVF 1899TGCGGAACATGGGATAACACCCTGAGTCTTTGGGTGTTC 1076 CGTWDNTLSAGVF 1900TGCGGAACATGGGATAACACCCTGAGTGCTGGGGTCTTC 1077 CGTWDNTLLTVLF 1901TGCGGAACATGGGACAACACTCTGCTTACTGTGTTATTC 1078 CGTWDNRLSSVIF 1902TGCGGAACATGGGATAACAGACTGAGTAGTGTGATTTTC 1079 CGTWDNRLSAVVF 1903TGCGGAACATGGGATAACAGGTTGAGTGCTGTGGTCTTC 1080 CGTWDNRLSAGGIF 1904TGCGGAACATGGGATAACAGGCTGAGTGCTGGTGGGATATTC 1081 CGTWDNRLSAEVF 1905TGCGGAACATGGGATAACAGACTGAGTGCTGAGGTGTTC 1082 CGTWDNRLRVGVL 1906TGTGGAACATGGGATAACAGACTGCGTGTTGGGGTTCTC 1083 CGTWDNRLLENVF 1907TGCGGAACATGGGATAATCGCCTGCTTGAGAATGTCTTC 1084 CGTWDNNLRAVF 1908TGCGGAACATGGGATAACAACCTGCGTGCTGTCTTC 1085 CGTWDNNLRAGVF 1909TGCGGAACTTGGGATAATAACCTGCGTGCTGGAGTGTTC 1086 CGTWDNNLGGGRVF 1910TGCGGAACATGGGACAACAATTTGGGCGGTGGCCGGGTGTTC 1087 CGTWDNNLGAGVL 1911TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCCTC 1088 CGTWDNNLGAGVF 1912TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCTTC 1089 CGTWDNILSAAVF 1913TGCGGAACTTGGGATAACATCCTGAGCGCTGCGGTGTTC 1090 CGTWDNILDAGVF 1914TGCGGAACCTGGGATAACATCTTGGATGCAGGGGTTTTC 1091 CGTWDNDLSGWLF 1915TGCGGAACATGGGATAACGACCTGAGTGGTTGGCTGTTC 1092 CGTWDNDLSAWVF 1916TGCGGAACATGGGATAACGACCTGAGTGCCTGGGTGTTC 1093 CGTWDLTLGGVVF 1917TGCGGAACATGGGATCTCACCCTGGGTGGTGTGGTGTTC 1094 CGTWDLSLSAGVF 1918TGCGGAACATGGGATCTCAGCCTGAGTGCTGGGGTATTC 1095 CGTWDLSLKEWVF 1919TGCGGAACATGGGATCTCAGCCTGAAAGAATGGGTGTTC 1096 CGTWDLSLDAVVF 1920TGCGGAACGTGGGATCTCAGCCTGGATGCTGTTGTTTTC 1097 CGTWDLKVF 1921TGCGGAACCTGGGACCTGAAGGTTTTC 1098 CGTWDKTLSVWVF 1922TGCGGAACATGGGATAAGACTCTGAGTGTTTGGGTGTTC 1099 CGTWDKSLSVWVF 1923TGCGGAACATGGGATAAGAGCCTGAGTGTTTGGGTGTTC 1100 CGTWDKSLSGVVF 1924TGCGGAACATGGGATAAGAGCCTGAGTGGTGTGGTATTT 1101 CGTWDKSLSDWVF 1925TGCGGAACATGGGATAAGAGCCTGAGTGATTGGGTGTTC 1102 CGTWDKSLSALVF 1926TGCGGAACATGGGATAAGAGCCTGAGTGCTTTGGTTTTC 1103 CGTWDKSLSAGVF 1927TGCGGAACATGGGATAAGAGCCTGAGTGCTGGCGTCTTC 1104 CGTWDKSLSADVF 1928TGCGGAACATGGGATAAGAGCCTGAGTGCCGACGTCTTC 1105 CGTWDKRLTIVVF 1929TGCGGAACATGGGATAAACGCCTGACTATTGTGGTCTTC 1106 CGTWDKRLSAWVL 1930TGCGGAACATGGGATAAACGCCTGAGTGCCTGGGTGCTC 1107 CGTWDKNLRAVVF 1931TGCGGAACATGGGATAAGAACCTGCGTGCTGTGGTCTTC 1108 CGTWDITLSGFVF 1932TGCGGAACATGGGATATCACCCTGAGTGGGTTTGTCTTC 1109 CGTWDITLHTGVF 1933TGCGGAACATGGGATATCACCTTGCATACTGGAGTATTC 1110 CGTWDISVTVVF 1934TGCGGAACATGGGATATCAGTGTGACTGTGGTGTTC 1111 CGTWDISVRGYAF 1935TGCGGAACATGGGATATCAGTGTGAGGGGTTATGCCTTC 1112 CGTWDISRWVF 1936TGCGGAACATGGGATATCAGCCGTTGGGTTTTC 1113 CGTWDISPSAWVF 1937TGCGGAACATGGGATATCAGCCCGAGTGCTTGGGTGTTC 1114 CGTWDISLSVWVF 1938TGCGGAACATGGGATATTAGCCTGAGTGTCTGGGTGTTC 1115 CGTWDISLSVVF 1939TGCGGAACATGGGATATCAGCCTGAGTGTTGTATTC 1116 CGTWDISLSSVVF 1940TGCGGAACTTGGGATATCAGCCTGAGTTCTGTGGTGTTC 1117 CGTWDISLSHWLF 1941TGCGGAACATGGGATATCAGCCTGAGTCACTGGTTGTTC 1118 CGTWDISLSGWVF 1942TGCGGAACATGGGATATCAGTCTGAGTGGTTGGGTGTTC 1119 CGTWDISLSGRVF 1943TGCGGAACATGGGATATCAGCCTGAGTGGTCGAGTGTTC 1120 CGTWDISLSAWAF 1944TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGCGTTC 1121 CGTWDISLSAVVF 1945TGCGGAACATGGGATATCAGCCTGAGTGCTGTGGTTTTC 1122 CGTWDISLSAVIF 1946TGCGGGACATGGGACATCAGCCTGAGTGCTGTGATATTC 1123 CGTWDISLSAVF 1947TGCGGAACATGGGATATCAGCCTGAGTGCTGTGTTC 1124 CGTWDISLSARVF 1948TGCGGAACATGGGATATCAGCCTGAGTGCCCGGGTGTTC 1125 CGTWDISLSALVF 1949TGCGGAACATGGGATATCAGCCTGAGTGCCCTGGTGTTC 1126 CGTWDISLSAHVF 1950TGCGGAACATGGGATATTAGCCTGAGTGCCCATGTCTTC 1127 CGTWDISLSAGVVF 1951TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGGTATTC 1128 CGTWDISLSAGPYVF 1952TGCGGAACATGGGATATCAGCCTGAGTGCCGGCCCTTATGTCTTC 1129 CGTWDISLSAGGVF 1953TGCGGCACATGGGATATCAGCCTGAGTGCTGGAGGGGTGTTC 1130 CGTWDISLSAEVF 1954TGCGGAACATGGGATATCAGCCTGAGTGCTGAGGTTTTC 1131 CGTWDISLSAAVF 1955TGCGGAACATGGGATATCAGCCTGAGTGCTGCTGTGTTC 1132 CGTWDISLRAVF 1956TGCGGAACATGGGATATCAGCCTGCGTGCTGTGTTC 1133 CGTWDISLNTGVF 1957TGCGGAACATGGGATATTAGCCTGAATACTGGGGTGTTC 1134 CGTWDISLNNYVF 1958TGCGGAACATGGGATATCAGCCTAAATAATTATGTCTTC 1135 CGTWDISLIAGVF 1959TGCGGAACATGGGATATCAGCCTAATTGCTGGGGTATTC 1136 CGTWDISLHTWLF 1960TGCGGAACATGGGATATCAGCCTGCATACTTGGCTGTTC 1137 CGTWDIRLTDELLF 1961TGCGGAACATGGGATATCCGCCTGACCGATGAGCTGTTATTC 1138 CGTWDIRLSGFVF 1962TGCGGAACATGGGATATCAGACTGAGCGGTTTTGTTTTC 1139 CGTWDINLGAGGLYVF 1963TGCGGAACATGGGATATCAACCTGGGTGCTGGGGGCCTTTATGTCTTC 1140 CGTWDIILSAEVF 1964TGCGGAACATGGGATATCATCCTGAGTGCTGAGGTATTC 1141 CGTWDHTLSAVF 1965TGCGGAACATGGGATCACACCCTGAGTGCTGTCTTC 1142 CGTWDHTLLTVLF 1966TGCGGAACATGGGACCACACTCTGCTTACTGTGTTATTC 1143 CGTWDHSLTAVVF 1967TGCGGAACATGGGATCACAGCCTGACTGCTGTGGTATTC 1144 CGTWDHSLTAGIF 1968TGCGGAACCTGGGATCACAGCCTGACTGCTGGGATATTC 1145 CGTWDHSLSVVLF 1969TGCGGAACATGGGATCACAGCCTGAGTGTTGTATTATTC 1146 CGTWDHSLSLVF 1970TGCGGAACATGGGATCACAGCCTGAGTTTGGTATTC 1147 CGTWDHSLSIGVF 1971TGCGGAACATGGGATCACAGCCTGTCTATTGGGGTTTTC 1148 CGTWDHSLSAGVF 1972TGCGGAACATGGGATCACAGCCTGAGTGCTGGGGTGTTC 1149 CGTWDHSLSAFVF 1973TGTGGAACTTGGGATCACAGCCTGAGTGCTTTCGTGTTC 1150 CGTWDHSLSAAVF 1974TGCGGAACATGGGATCACAGTCTGAGTGCTGCTGTTTTC 1151 CGTWDHNLRAVF 1975TGCGGAACATGGGACCACAATCTGCGTGCTGTCTTC 1152 CGTWDFTLSVGRF 1976TGCGGGACATGGGATTTCACCCTGAGTGTTGGGCGCTTC 1153 CGTWDFTLSAPVF 1977TGCGGAACATGGGATTTCACCCTGAGTGCTCCTGTCTTC 1154 CGTWDFSVSAGWVF 1978TGCGGAACGTGGGATTTCAGCGTGAGTGCTGGGTGGGTGTTC 1155 CGTWDFSLTTWLF 1979TGCGGAACGTGGGATTTCAGTCTTACTACCTGGTTATTC 1156 CGTWDFSLSVWVF 1980TGCGGAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1157 CGTWDFSLSTGVF 1981TGCGGAACATGGGATTTCAGCCTGAGTACTGGGGTTTTC 1158 CGTWDFSLSGVVF 1982TGCGGCACATGGGATTTCAGCCTGAGTGGTGTGGTATTC 1159 CGTWDFSLSGFVF 1983TGCGGAACATGGGATTTCAGCCTGAGTGGTTTCGTGTTC 1160 CGTWDFSLSAGVF 1984TGCGGAACATGGGATTTCAGCCTGAGTGCTGGGGTGTTC 1161 CGTWDETVRGWVF 1985TGCGGAACATGGGATGAAACCGTGAGAGGTTGGGTGTTC 1162 CGTWDESLRSWVF 1986TGCGGAACATGGGATGAAAGTCTGAGAAGCTGGGTGTTC 1163 CGTWDERQTDESYVF 1987TGCGGAACTTGGGATGAGAGGCAGACTGATGAGTCCTATGTCTTC 1164 CGTWDERLVAGQVF 1988TGCGGAACATGGGATGAGAGACTCGTTGCTGGCCAGGTCTTC 1165 CGTWDERLSPGAFF 1989TGCGGAACATGGGATGAGAGACTGAGTCCTGGAGCTTTTTTC 1166 CGTWDEKVF 1990TGCGGAACATGGGATGAGAAGGTGTTC 1167 CGTWDEGQTTDFFVF 1991TGCGGAACCTGGGATGAAGGCCAGACTACTGATTTCTTTGTCTTC 1168 CGTWDDTLAGVVF 1992TGCGGAACATGGGATGACACCCTGGCTGGTGTGGTCTTC 1169 CGTWDDRLTSAVF 1993TGCGGAACATGGGATGACAGGCTGACTTCTGCGGTCTTC 1170 CGTWDDRLFVVVF 1994TGCGGAACATGGGATGACAGACTGTTTGTTGTGGTATTC 1171 CGTWDDNLRGWVF 1995TGCGGAACATGGGATGATAACCTGAGAGGTTGGGTGTTC 1172 CGTWDDNLRGVVF 1996TGCGGAACATGGGATGACAACCTGCGTGGTGTCGTGTTC 1173 CGTWDDNLNIGRVF 1997TGCGGAACCTGGGATGACAATTTGAATATTGGAAGGGTGTTC 1174 CGTWDDILSAVIF 1998TGCGGAACATGGGATGACATCCTGAGTGCTGTGATATTC 1175 CGTWDDILRGWVF 1999TGCGGAACATGGGATGATATCCTGAGAGGTTGGGTGTTC 1176 CGTWDATLSPGWLF 2000TGCGGAACATGGGATGCCACCCTGAGTCCTGGGTGGTTATTC 1177 CGTWDASVTSWVF 2001TGCGGAACATGGGATGCCAGCGTGACTTCTTGGGTGTTC 1178 CGTWDASLTSVVF 2002TGCGGAACATGGGATGCCAGCCTGACTTCTGTGGTCTTC 1179 CGTWDASLSVWVF 2003TGCGGAACATGGGATGCCAGCCTGAGTGTTTGGGTGTTC 1180 CGTWDASLSVPWVF 2004TGCGGAACATGGGATGCCAGCCTGAGTGTTCCTTGGGTGTTC 1181 CGTWDASLSVAVF 2005TGCGGAACATGGGATGCCAGCCTGAGTGTGGCGGTATTC 1182 CGTWDASLSTWVF 2006TGCGGAACATGGGATGCCAGCCTGAGTACCTGGGTATTC 1183 CGTWDASLSGVVF 2007TGCGGAACATGGGATGCCAGCCTGAGTGGTGTGGTATTC 1184 CGTWDASLSGGGEF 2008TGCGGAACATGGGATGCCAGCCTGAGTGGTGGGGGAGAATTC 1185 CGTWDASLSAGVF 2009TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1186 CGTWDASLSAGLF 2010TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGCTTTTC 1187 CGTWDASLSAEVF 2011TGTGGCACATGGGATGCCAGCCTGAGTGCTGAAGTCTTC 1188 CGTWDASLSADFWVF 2012TGCGGAACATGGGATGCCAGCCTGAGTGCTGACTTTTGGGTGTTC 1189 CGTWDASLRVFF 2013TGCGGAACATGGGATGCCAGCCTGAGAGTCTTCTTC 1190 CGTWDASLRAVVL 2014TGCGGAACATGGGATGCCAGTCTGAGGGCTGTGGTACTC 1191 CGTWDASLNIWVF 2015TGCGGAACATGGGATGCCAGCCTGAATATTTGGGTTTTC 1192 CGTWDASLKNLVF 2016TGCGGGACATGGGATGCCAGCCTGAAGAATCTGGTCTTC 1193 CGTWDASLGAWVF 2017TGCGGAACATGGGATGCCAGCCTGGGTGCCTGGGTATTC 1194 CGTWDASLGAVVF 2018TGCGGAACATGGGATGCCAGCCTGGGTGCTGTGGTCTTC 1195 CGTWDASLGAGVF 2019TGCGGAACATGGGATGCCAGCCTGGGTGCGGGGGTCTTC 1196 CGTWDARLSGLYVF 2020TGCGGAACATGGGATGCTAGGCTGAGTGGCCTTTATGTCTTC 1197 CGTWDARLGGAVF 2021TGTGGAACCTGGGATGCGAGACTGGGTGGTGCAGTCTTC 1198 CGTWDANLRAGVF 2022TGCGGAACATGGGATGCCAATCTGCGTGCTGGGGTCTTC 1199 CGTWDAIISGWVF 2023TGCGGAACATGGGATGCTATCATAAGTGGTTGGGTGTTC 1200 CGTWDAGQSVWVF 2024TGCGGAACATGGGATGCCGGCCAGAGTGTTTGGGTGTTC 1201 CGTWDAGLTGLYVF 2025TGCGGCACATGGGATGCCGGGCTGACTGGCCTTTATGTCTTC 1202 CGTWDAGLSVYVF 2026TGCGGAACTTGGGATGCCGGTCTGAGTGTTTATGTCTTC 1203 CGTWDAGLSTGVF 2027TGCGGGACATGGGATGCCGGCCTGAGTACTGGGGTCTTC 1204 CGTWDAGLSGDVF 2028TGCGGAACATGGGATGCCGGCCTGAGTGGGGACGTTTTC 1205 CGTWDAGLSAGYVF 2029TGCGGAACATGGGATGCCGGCCTGAGTGCTGGTTATGTCTTC 1206 CGTWDAGLRVWVF 2030TGCGGAACATGGGATGCCGGCCTGCGTGTTTGGGTGTTC 1207 CGTWDAGLREIF 2031TGCGGAACATGGGATGCCGGCCTGAGGGAAATTTTC 1208 CGTWASSLSSWVF 2032TGCGGAACATGGGCCAGCAGCCTGAGTTCTTGGGTGTTC 1209 CGTWAGSLSGHVF 2033TGCGGAACATGGGCTGGCAGCCTGAGTGGTCATGTCTTC 1210 CGTWAGSLSAAWVF 2034TGCGGAACATGGGCTGGCAGCCTGAGTGCCGCTTGGGTGTTC 1211 CGTWAGSLNVYWVF 2035TGCGGAACATGGGCTGGCAGCCTGAATGTTTATTGGGTGTTC 1212 CGTWAGNLRPNWVF 2036TGCGGAACATGGGCTGGCAACCTGAGACCTAATTGGGTGTTC 1213 CGTRGSLGGAVF 2037TGCGGAACAAGGGGTAGCCTGGGTGGTGCGGTGTTC 1214 CGTRDTTLSVPVF 2038TGCGGAACAAGGGATACCACCCTGAGTGTCCCGGTGTTC 1215 CGTRDTSLNIEIF 2039TGCGGAACACGGGATACCAGCCTCAATATTGAAATCTTC 1216 CGTRDTSLNDVF 2040TGTGGAACACGGGATACCAGCCTGAATGATGTCTTC 1217 CGTRDTRLSIVVF 2041TGCGGAACACGGGATACCCGCCTGAGTATTGTGGTTTTC 1218 CGTRDTILSAEVF 2042TGCGGCACACGGGATACCATCCTGAGTGCTGAGGTGTTC 1219 CGTRDRSLSGWVF 2043TGCGGAACACGGGATAGAAGCCTGAGTGGTTGGGTGTTC 1220 CGSWYYNVFLF 2044TGCGGATCATGGTATTACAATGTCTTCCTTTTC 1221 CGSWHSSLNLVVF 2045TGCGGATCTTGGCATAGCAGCCTCAACCTTGTCGTCTTC 1222 CGSWGSGLSAPYVF 2046TGCGGATCATGGGGTAGTGGCCTGAGTGCCCCTTATGTCTTC 1223 CGSWESGLGAWLF 2047TGCGGTTCGTGGGAAAGCGGCCTGGGTGCTTGGCTGTTC 1224 CGSWDYGLLLF 2048TGCGGATCCTGGGATTACGGCCTCCTACTCTTC 1225 CGSWDVSLTAVF 2049TGCGGTTCATGGGATGTCAGCCTGACTGCTGTTTTC 1226 CGSWDVSLNVGIF 2050TGCGGATCCTGGGATGTCAGTCTCAATGTTGGCATTTTC 1227 CGSWDTTLRAWVF 2051TGCGGATCATGGGATACCACCCTGCGTGCTTGGGTGTTC 1228 CGSWDTSPVRAWVF 2052TGCGGCTCGTGGGATACCAGCCCTGTCCGTGCTTGGGTGTTC 1229 CGSWDTSLSVWVF 2053TGCGGATCATGGGATACCAGCCTGAGTGTTTGGGTGTTC 1230 CGSWDTSLSAEVF 2054TGCGGATCATGGGATACCAGCCTGAGTGCTGAGGTGTTC 1231 CGSWDTSLRAWVF 2055TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGTGTTC 1232 CGSWDTSLRAWAF 2056TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGCGTTC 1233 CGSWDTSLDARLF 2057TGCGGATCATGGGATACCAGCCTGGATGCTAGGCTGTTC 1234 CGSWDTILLVYVF 2058TGCGGATCATGGGATACCATCCTGCTTGTCTATGTCTTC 1235 CGSWDRWQAAVF 2059TGCGGATCATGGGATCGCTGGCAGGCTGCTGTCTTC 1236 CGSWDRSLSGYVF 2060TGCGGATCATGGGATAGGAGCCTGAGTGGGTATGTCTTC 1237 CGSWDRSLSAYVF 2061TGCGGATCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 1238 CGSWDRSLSAVVF 2062TGCGGATCATGGGATAGGAGCCTGAGTGCCGTGGTTTTC 1239 CGSWDNTLGVVLF 2063TGCGGATCATGGGATAACACCTTGGGTGTTGTTCTCTTC 1240 CGSWDNRLSTVIF 2064TGCGGATCGTGGGATAACAGACTAAGTACTGTCATCTTC 1241 CGSWDNRLNTVIF 2065TGCGGAAGCTGGGATAATCGATTGAACACTGTGATTTTC 1242 CGSWDLSPVRVLVF 2066TGCGGTTCATGGGATCTCAGCCCTGTACGTGTCCTTGTGTTC 1243 CGSWDLSLSAVVF 2067TGCGGATCATGGGATCTCAGCCTGAGTGCTGTCGTTTTC 1244 CGSWDKNLRAVLF 2068TGCGGATCATGGGATAAAAACCTGCGTGCTGTGCTGTTC 1245 CGSWDISLSAGVF 2069TGCGGCTCATGGGATATCAGCCTGAGTGCTGGGGTGTTC 1246 CGSWDIRLSAEVF 2070TGCGGATCATGGGATATCAGACTGAGTGCAGAGGTCTTC 1247 CGSWDIKLNIGVF 2071TGCGGATCATGGGACATCAAACTGAATATTGGGGTATTC 1248 CGSWDFSLNYFVF 2072TGCGGATCATGGGATTTCAGTCTCAATTATTTTGTCTTC 1249 CGSWDASLSIEVF 2073TGCGGATCATGGGATGCCAGCCTGAGTACTGAGGTGTTC 1250 CGSWDAGLRGWVF 2074TGCGGATCCTGGGATGCCGGCCTGCGTGGCTGGGTTTTC 1251 CGRWESSLGAVVF 2075TGCGGAAGATGGGAGAGCAGCCTGGGTGCTGTGGTTTTC 1252 CGRWDFSLSAYVF 2076TGCGGAAGATGGGATTTTAGTCTGAGTGCTTATGTCTTC 1253 CGQWDNDLSVWVF 2077TGCGGACAATGGGATAACGACCTGAGTGTTTGGGTGTTC 1254 CGPWHSSVTSGHVL 2078TGCGGACCCTGGCATAGCAGCGTGACTAGTGGCCACGTGCTC 1255 CGLWDASLSAPTWVF 2079TGCGGATTATGGGATGCCAGCCTGAGTGCTCCTACTTGGGTGTTC 1256 CGIWHTSLSAWVF 2080TGTGGAATATGGCACACTAGCCTGAGTGCTTGGGTGTTC 1257 CGIWDYSLDTWVF 2081TGCGGAATATGGGATTACAGCCTGGATACTTGGGTGTTC 1258 CGIWDTSLSAWVF 2082TGCGGCATATGGGATACCAGCCTGAGTGCTTGGGTGTTC 1259 CGIWDTRLSVYVF 2083TGCGGAATTTGGGATACCAGGCTGAGTGTTTATGTCTTC 1260 CGIWDTRLSVYIF 2084TGCGGAATTTGGGATACCAGGCTGAGTGTTTATATCTTC 1261 CGIWDTNLGYLF 2085TGTGGAATATGGGATACGAATCTGGGTTATCTCTTC 1262 CGIWDTGLSAVVF 2086TGCGGTATATGGGATACCGGCCTGAGTGCTGTGGTATTC 1263 CGIWDRSLSAWVF 2087TGCGGAATATGGGATCGCAGCCTGAGTGCTTGGGTGTTT 1264 CGIRDTRLSVYVF 2088TGCGGAATTCGGGATACCAGGCTGAGTGTTTATGTCTTC 1265 CGGWSSRLGVGPVF 2089TGCGGAGGATGGAGTAGCAGACTGGGTGTTGGCCCAGTGTTT 1266 CGGWGSGLSAWVF 2090TGCGGAGGATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 1267 CGGWDTSLSAWVF 2091TGCGGAGGATGGGATACCAGCCTGAGTGCTTGGGTGTTC 1268 CGGWDRGLDAWVF 2092TGCGGAGGATGGGATAGGGGCCTGGATGCTTGGGTTTTC 1269 CGAWRNNVWVF 2093TGCGGAGCATGGCGTAATAACGTGTGGGTGTTC 1270 CGAWNRRLNPHSHWVF 2094TGCGGAGCATGGAACAGGCGCCTGAATCCTCATTCTCATTGGGTGTTC 1271 CGAWHNKLSAVF 2095TGCGGAGCCTGGCACAACAAACTGAGCGCGGTCTTC 1272 CGAWGSSLRASVF 2096TGCGGAGCATGGGGTAGCAGCCTGAGAGCTAGTGTCTTC 1273 CGAWGSGLSAWVF 2097TGCGGAGCATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 1274 CGAWESSLSAPYVF 2098TGCGGAGCATGGGAAAGTAGCCTGAGTGCCCCTTATGTCTTC 1275 CGAWESSLNVGLI 2099TGCGGAGCATGGGAGAGCAGCCTCAATGTTGGACTGATC 1276 CGAWESGRSAGVVF 2100TGCGGAGCATGGGAGAGCGGCCGGAGTGCTGGGGTGGTGTTC 1277 CGAWDYSVSGWVF 2101TGCGGAGCTTGGGATTACAGTGTGAGTGGTTGGGTGTTC 1278 CGAWDYSLTAGVF 2102TGCGGAGCATGGGATTACAGCCTGACTGCCGGAGTATTC 1279 CGAWDYRLSAVLF 2103TGCGGAGCCTGGGATTACAGACTGAGTGCCGTGCTATTC 1280 CGAWDVRLDVGVF 2104TGCGGAGCGTGGGATGTTCGTCTGGATGTTGGGGTGTTC 1281 CGAWDTYSYVF 2105TGCGGAGCATGGGATACCTACAGTTATGTCTTC 1282 CGAWDTTLSGVVF 2106TGCGGAGCATGGGATACGACCCTGAGTGGTGTGGTATTC 1283 CGAWDTTLSAVIF 2107TGCGGAGCGTGGGATACTACCCTGAGTGCTGTGATATTC 1284 CGAWDTSQGASYVF 2108TGCGGCGCATGGGATACCAGCCAGGGTGCGTCTTATGTCTTT 1285 CGAWDTSPVRAGVF 2109TGCGGAGCATGGGATACCAGCCCTGTACGTGCTGGGGTGTTC 1286 CGAWDTSLWLF 2110TGCGGAGCATGGGATACCAGCCTGTGGCTTTTC 1287 CGAWDTSLTVYVF 2111TGCGGAGCATGGGATACCAGCCTGACTGTTTATGTCTTC 1288 CGAWDTSLTAGVF 2112TGCGGAGCATGGGACACCAGTCTGACTGCTGGGGTGTTC 1289 CGAWDTSLSTVVF 2113TGCGGAGCTTGGGATACCAGCCTGAGTACTGTGGTTTTC 1290 CGAWDTSLSSRYIF 2114TGCGGAGCATGGGATACCAGCCTGAGTTCTAGATACATATTC 1291 CGAWDTSLSGYVF 2115TGCGGAGCATGGGATACCAGCCTGAGTGGTTATGTCTTC 1292 CGAWDTSLSGWVF 2116TGCGGAGCCTGGGATACCAGCCTGAGTGGCTGGGTGTTC 1293 CGAWDTSLSGVLF 2117TGCGGAGCATGGGATACCAGTCTGAGTGGTGTGCTATTC 1294 CGAWDTSLSGLVF 2118TGCGGAGCTTGGGATACCAGCTTGAGTGGTCTTGTTTTC 1295 CGAWDTSLSGFVF 2119TGCGGAGCTTGGGATACCAGCTTGAGTGGTTTTGTTTTC 1296 CGAWDTSLSGEVF 2120TGCGGAGCATGGGATACCAGCCTGAGTGGTGAGGTCTTT 1297 CGAWDTSLSDFVF 2121TGCGGAGCTTGGGATACCAGCTTGAGTGATTTTGTTTTC 1298 CGAWDTSLRTAIF 2122TGCGGAGCATGGGATACCAGCCTGCGAACTGCGATATTC 1299 CGAWDTSLRLF 2123TGCGGAGCATGGGATACCAGCCTGCGGCTTTTC 1300 CGAWDTSLNVHVF 2124TGCGGAGCATGGGATACCAGCCTGAATGTTCATGTCTTC 1301 CGAWDTSLNKWVF 2125TGCGGAGCATGGGATACCAGCCTCAATAAATGGGTGTTC 1302 CGAWDTRLSARLF 2126TGCGGAGCATGGGATACCCGCCTCAGTGCGCGGCTGTTC 1303 CGAWDTRLRGFIF 2127TGCGGAGCATGGGATACCAGACTGAGGGGTTTTATTTTC 1304 CGAWDTNLGNVLL 2128TGCGGAGCATGGGATACTAATTTGGGGAATGTTCTCCTC 1305 CGAWDTNLGKWVF 2129TGCGGGGCATGGGATACCAACCTGGGTAAATGGGTTTTC 1306 CGAWDTGLEWYVF 2130TGCGGAGCATGGGATACCGGCCTTGAGTGGTATGTTTTT 1307 CGAWDRTSGLWLF 2131TGCGGAGCATGGGATAGGACTTCTGGATTGTGGCTTTTC 1308 CGAWDRSLVAGLF 2132TGCGGAGCGTGGGATCGTAGCCTGGTTGCTGGACTCTTC 1309 CGAWDRSLTVYVF 2133TGCGGAGCGTGGGATAGAAGCCTGACTGTTTATGTCTTC 1310 CGAWDRSLSGYVF 2134TGCGGAGCATGGGATAGAAGCCTGAGTGGTTATGTCTTC 1311 CGAWDRSLSAYVF 2135TGCGGAGCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 1312 CGAWDRSLSAVVF 2136TGCGGAGCATGGGATAGAAGCCTGAGTGCGGTGGTATTC 1313 CGAWDRSLSAGVF 2137TGCGGAGCATGGGATCGCAGCCTGAGTGCTGGGGTTTTC 1314 CGAWDRSLRIVVF 2138TGCGGAGCGTGGGATCGCAGCCTGCGTATTGTGGTATTC 1315 CGAWDRSLRAYVF 2139TGCGGAGCATGGGATAGAAGTCTGAGGGCTTACGTCTTC 1316 CGAWDRSLNVWLF 2140TGCGGAGCATGGGATAGAAGTCTGAATGTTTGGCTGTTC 1317 CGAWDRGLNVGWLF 2141TGCGGCGCCTGGGATAGGGGCCTGAATGTCGGTTGGCTTTTC 1318 CGAWDNRLSILAF 2142TGCGGCGCATGGGATAATAGACTGAGTATTTTGGCCTTC 1319 CGAWDNDLTAYVF 2143TGCGGAGCTTGGGATAATGACCTGACAGCTTATGTCTTC 1320 CGAWDFSLTPLF 2144TGCGGGGCATGGGATTTCAGCCTGACTCCTCTCTTC 1321 CGAWDDYRGVSIYVF 2145TGCGGAGCCTGGGATGACTATCGGGGTGTGAGTATTTATGTCTTC 1322 CGAWDDRPSSAVVF 2146TGTGGAGCATGGGATGACCGGCCTTCGAGTGCCGTGGTTTTC 1323 CGAWDDRLTVVVF 2147TGCGGAGCATGGGATGACAGACTGACTGTCGTTGTTTTC 1324 CGAWDDRLGAVF 2148TGCGGAGCGTGGGATGACAGGCTGGGTGCTGTGTTC 1325 CGAWDASLNPGRAF 2149TGCGGAGCGTGGGATGCCAGCCTGAATCCTGGCCGGGCATTC 1326 CGAWDAGLRE1F 2150TGCGGAGCATGGGATGCCGGCCTGAGGGAAATTTTC 1327 CGAWAGSPSPWVF 2151TGCGGAGCTTGGGCTGGCAGTCCGAGTCCTTGGGTTTTC 1328 CGAFDTTLSAGVF 2152TGCGGAGCATTCGACACCACCCTGAGTGCTGGCGTTTTC 1329 CETWESSLSVGVF 2153TGCGAAACATGGGAGAGCAGCCTGAGTGTTGGGGTCTTC 1330 CETWESSLRVWVF 2154TGCGAAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC 1331 CETWDTSLSGGVF 2155TGCGAAACGTGGGATACCAGCCTGAGTGGTGGGGTGTTC 1332 CETWDTSLSDFYVF 2156TGCGAAACATGGGATACCAGCCTGAGTGACTTTTATGTCTTC 1333 CETWDTSLSALF 2157TGCGAAACATGGGATACCAGCCTGAGTGCCCTCTTC 1334 CETWDTSLRAEVF 2158TGCGAAACATGGGATACCAGCCTGCGTGCTGAAGTCTTC 1335 CETWDTSLNVVVF 2159TGCGAAACATGGGATACCAGCCTGAATGTTGTGGTATTC 1336 CETWDTSLGAVVF 2160TGCGAAACATGGGATACCAGCCTGGGTGCCGTGGTGTTC 1337 CETWDRSLSGVVF 2161TGCGAAACATGGGATAGAAGCCTGAGTGGTGTGGTATTC 1338 CETWDRSLSAWVF 2162TGCGAAACATGGGATAGGAGCCTGAGTGCTTGGGTGTTT 1339 CETWDRSLSAVVF 2163TGCGAAACATGGGATCGCAGCCTGAGTGCTGTGGTCTTC 1340 CETWDRGLSVVVF 2164TGCGAGACGTGGGATAGAGGCCTGAGTGTTGTGGTTTTC 1341 CETWDRGLSAVVF 2165TGCGAAACATGGGATAGGGGCCTGAGTGCAGTGGTATTC 1342 CETWDHTLSVVIF 2166TGCGAAACATGGGATCACACCCTGAGTGTTGTGATATTC 1343 CETWDASLTVVLF 2167TGCGAAACATGGGATGCCAGCCTGACTGTTGTGTTATTC 1344 CETWDASLSAGVF 2168TGCGAAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1345 CETWDAGLSEVVF 2169TGCGAAACGTGGGATGCCGGCCTGAGTGAGGTGGTGTTC 1346 CETFDTSLSVVVF 2170TGCGAAACATTTGATACCAGCCTGAGTGTTGTAGTCTTC 1347 CETFDTSLNIVVF 2171TGCGAAACATTTGATACCAGCCTAAATATTGTAGTCTTT 1348 CESWDRSRIGVVF 2172TGCGAATCATGGGATAGAAGCCGGATTGGTGTGGTCTTC 1349 CESWDRSLSARVY 2173TGCGAAAGTTGGGACAGGAGTCTGAGTGCCCGGGTGTAC 1350 CESWDRSLRAVVF 2174TGCGAATCCTGGGATAGGAGCCTGCGTGCCGTGGTCTTC 1351 CESWDRSLIVVF 2175TGCGAATCTTGGGATCGTAGTTTGATTGTGGTGTTC 1352 CESWDNNLNEVVF 2176TGCGAAAGTTGGGATAACAATTTAAATGAGGTGGTTTTC 1353 CEIWESSPSADDLVF 2177TGCGAAATATGGGAGAGCAGCCCGAGTGCTGACGATTTGGTGTTC 1354 CEAWDTSLSGAVF 2178TGCGAAGCATGGGATACCAGCCTGAGTGGTGCGGTGTTC 1355 CEAWDTSLSAGVF 2179TGCGAAGCATGGGATACCAGCCTGAGTGCCGGGGTGTTC 1356 CEAWDTSLGGGVF 2180TGCGAAGCATGGGATACCAGCCTGGGTGGTGGGGTGTTC 1357 CEAWDRSLTGSLF 2181TGCGAAGCATGGGATCGCAGCCTGACTGGTAGCCTGTTC 1358 CEAWDRGLSAVVF 2182TGCGAAGCGTGGGATAGGGGCCTGAGTGCAGTGGTATTC 1359 CEAWDNILSTVVF 2183TGCGAAGCCTGGGATAACATCCTGAGTACTGTGGTGTTC 1360 CEAWDISLSAGVF 2184TGCGAAGCATGGGACATCAGCCTGAGTGCTGGGGTGTTC 1361 CEAWDADLSGAVF 2185TGCGAAGCATGGGATGCCGACCTGAGTGGTGCGGTGTTC 1362 CATWTGSFRTGHYVF 2186TGCGCAACATGGACTGGTAGTTTCAGAACTGGCCATTATGTCTTC 1363 CATWSSSPRGWVF 2187TGCGCAACATGGAGTAGCAGTCCCAGGGGGTGGGTGTTC 1364 CATWHYSLSAGRVF 2188TGCGCAACATGGCATTACAGCCTGAGTGCTGGCCGAGTGTTC 1365 CATWHTSLSIVQF 2189TGCGCAACATGGCATACCAGCCTGAGTATTGTGCAGTTC 1366 CATWHSTLSADVLF 2190TGCGCAACATGGCATAGCACCCTGAGTGCTGATGTGCTTTTC 1367 CATWHSSLSAGRLF 2191TGCGCAACATGGCATAGCAGCCTGAGTGCTGGCCGACTCTTC 1368 CATWHIARSAWVF 2192TGCGCAACATGGCATATCGCTCGGAGTGCCTGGGTGTTC 1369 CATWGSSQSAVVF 2193TGCGCAACATGGGGTAGTAGTCAGAGTGCCGTGGTATTC 1370 CATWGSSLSAGGVF 2194TGCGCAACATGGGGTAGCAGCCTGAGTGCTGGGGGTGTTTTC 1371 CATWEYSLSVVLF 2195TGTGCAACATGGGAATACAGCCTGAGTGTTGTGCTGTTC 1372 CATWETTRRASFVF 2196TGCGCAACATGGGAGACCACCCGACGTGCCTCTTTTGTCTTC 1373 CATWETSLNVYVF 2197TGCGCAACATGGGAGACCAGCCTGAATGTTTATGTCTTC 1374 CATWETSLNVVVF 2198TGCGCAACATGGGAAACTAGCCTGAATGTTGTGGTCTTC 1375 CATWETSLNLYVF 2199TGCGCAACATGGGAGACCAGCCTGAATCTTTATGTCTTC 1376 CATWETGLSAGEVF 2200TGCGCAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC 1377 CATWESTLSVVVF 2201TGCGCGACGTGGGAGAGTACCCTAAGTGTTGTGGTTTTC 1378 CATWESSLSIFVF 2202TGCGCAACGTGGGAGAGCAGCCTGAGTATTTTTGTCTTC 1379 CATWESSLNTFYVF 2203TGCGCAACATGGGAAAGCAGCCTCAACACTTTTTATGTCTTC 1380 CATWESRVDTRGLLF 2204TGCGCAACATGGGAGAGTAGGGTGGATACTCGAGGGTTGTTATTC 1381 CATWESGLSGAGVF 2205TGCGCAACATGGGAGAGCGGCCTGAGTGGTGCGGGGGTGTTC 1382 CATWEGSLNTFYVF 2206TGCGCAACATGGGAAGGCAGCCTCAACACTTTTTATGTCTTC 1383 CATWDYSLSAVVF 2207TGCGCAACTTGGGATTATAGCCTGAGTGCTGTGGTGTTC 1384 CATWDYRLSIVVF 2208TGCGCAACATGGGATTACAGACTGAGTATTGTGGTATTC 1385 CATWDYNLGAAVF 2209TGCGCAACATGGGATTATAACCTGGGAGCTGCGGTGTTC 1386 CATWDVTLGVLHF 2210TGCGCCACATGGGATGTCACCCTGGGTGTCTTGCATTTC 1387 CATWDTTLSVWVF 2211TGCGCAACATGGGATACAACACTGAGTGTCTGGGTCTTC 1388 CATWDTTLSVVLF 2212TGCGCAACATGGGATACCACCCTGAGTGTAGTACTTTTC 1389 CATWDTTLSVEVF 2213TGCGCAACATGGGATACCACCCTGAGTGTTGAGGTCTTC 1390 CATWDTSPSLSGFWVF 2214TGCGCAACATGGGATACCAGCCCCAGCCTGAGTGGTTTTTGGGTGTTC 1391 CATWDTSLTGVVF 2215TGCGCAACATGGGATACCAGCCTGACTGGTGTGGTATTC 1392 CATWDTSLTGAVF 2216TGCGCAACATGGGATACCAGCCTGACTGGTGCGGTGTTC 1393 CATWDTSLTAWVF 2217TGCGCAACATGGGATACCAGCCTGACTGCCTGGGTATTC 1394 CATWDTSLTAVVF 2218TGCGCAACATGGGATACCAGCCTGACTGCTGTGGTTTTC 1395 CATWDTSLTAKVF 2219TGCGCAACATGGGATACTAGCCTGACTGCTAAGGTGTTC 1396 CATWDTSLSVVVF 2220TGCGCAACATGGGACACCAGCCTGAGTGTTGTGGTTTTC 1397 CATWDTSLSVGVF 2221TGCGCTACTTGGGATACCAGCCTGAGTGTTGGGGTATTT 1398 CATWDTSLSSWVF 2222TGCGCAACATGGGATACCAGCCTGAGTTCTTGGGTGTTC 1399 CATWDTSLSGGVL 2223TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTACTC 1400 CATWDTSLSGGVF 2224TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC 1401 CATWDTSLSGGRVF 2225TGCGCAACATGGGATACCAGCCTGAGTGGTGGCCGAGTGTTC 1402 CATWDTSLSGDRVF 2226TGCGCAACATGGGATACCAGCCTGAGTGGTGACCGAGTGTTC 1403 CATWDTSLSEGVF 2227TGCGCAACGTGGGATACTAGCCTGAGTGAAGGGGTGTTC 1404 CATWDTSLSAVVL 2228TGCGCAACCTGGGATACCAGCCTGAGTGCCGTGGTGCTC 1405 CATWDTSLSAVF 2229TGCGCAACATGGGATACCAGCCTGAGTGCTGTCTTC 1406 CATWDTSLSARVF 2230TGCGCGACATGGGATACCAGCCTGAGTGCTCGGGTGTTC 1407 CATWDTSLSALF 2231TGCGCAACATGGGATACCAGCCTGAGTGCCTTATTC 1408 CATWDTSLSAHVF 2232TGCGCAACATGGGATACCAGCCTGAGTGCTCATGTCTTC 1409 CATWDTSLSAGRVF 2233TGCGCAACATGGGATACCAGCCTGAGTGCTGGCCGGGTGTTC 1410 CATWDTSLSAEVF 2234TGCGCAACATGGGATACCAGCCTGAGTGCGGAGGTCTTC 1411 CATWDTSLSADAGGGVF 2235TGCGCAACATGGGATACCAGCCTGAGTGCTGATGCTGGTGGGGGGGTCTTC 1412 CATWDTSLRVVVF2236 TGCGCAACATGGGATACCAGCCTGCGTGTCGTGGTATTC 1413 CATWDTSLRGVF 2237TGCGCAACATGGGATACCAGCCTGAGAGGGGTGTTC 1414 CATWDTSLPAWVF 2238TGCGCAACATGGGATACCAGCCTGCCTGCGTGGGTGTTC 1415 CATWDTSLNVGVF 2239TGTGCAACATGGGATACCAGCCTGAATGTTGGGGTATTC 1416 CATWDTSLGIVLF 2240TGCGCAACATGGGATACCAGCCTGGGTATTGTGTTATTT 1417 CATWDTSLGARVVF 2241TGCGCAACATGGGACACCAGCCTGGGTGCGCGTGTGGTCTTC 1418 CATWDTSLGALF 2242TGTGCAACGTGGGATACCAGTCTAGGTGCCTTGTTC 1419 CATWDTSLATGLF 2243TGCGCAACATGGGATACCAGCCTGGCGACTGGACTGTTC 1420 CATWDTSLAAWVF 2244TGCGCAACATGGGATACCAGCCTGGCTGCCTGGGTATTC 1421 CATWDTRLSAVVF 2245TGCGCAACCTGGGATACCAGGCTGAGTGCTGTGGTCTTC 1422 CATWDTRLSAGVF 2246TGCGCAACATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1423 CATWDTRLLITVF 2247TGTGCAACGTGGGACACACGTCTACTTATTACGGTTTTC 1424 CATWDTLLSVELF 2248TGCGCAACATGGGACACCCTCCTGAGTGTTGAACTCTTC 1425 CATWDTGRNPHVVF 2249TGCGCAACATGGGATACTGGCCGCAATCCTCATGTGGTCTTC 1426 CATWDTGLSSVLF 2250TGCGCAACATGGGATACCGGCCTGTCTTCGGTGTTGTTC 1427 CATWDTGLSAVF 2251TGCGCAACGTGGGATACCGGCCTGAGTGCGGTTTTC 1428 CATWDRTLSIGVF 2252TGCGCTACGTGGGATAGGACCCTGAGTATTGGAGTCTTC 1429 CATWDRSVTAVLF 2253TGCGCAACGTGGGATCGCAGTGTGACTGCTGTGCTCTTC 1430 CATWDRSLSGVVF 2254TGCGCAACCTGGGATAGGAGCCTGAGTGGTGTGGTGTTC 1431 CATWDRSLSAVVF 2255TGCGCAACATGGGATAGAAGCCTGAGTGCTGTGGTCTTC 1432 CATWDRSLSAVPWVF 2256TGCGCAACATGGGATAGAAGCCTGAGTGCTGTTCCTTGGGTGTTC 1433 CATWDRSLSAGVF 2257TGCGCAACATGGGATCGCAGCCTGAGTGCTGGGGTGTTC 1434 CATWDRSLRAGVF 2258TGCGCAACGTGGGATAGGAGCCTGCGTGCTGGGGTGTTC 1435 CATWDRSLNVYVL 2259TGCGCAACATGGGATCGCAGTCTGAATGTTTATGTCCTC 1436 CATWDRILSAEVF 2260TGCGCAACGTGGGATCGCATCCTGAGCGCTGAGGTGTTC 1437 CATWDRGLSTGVF 2261TGCGCAACGTGGGATAGAGGCCTGAGTACTGGGGTGTTC 1438 CATWDNYLGAAVF 2262TGCGCAACATGGGATAACTACCTGGGTGCTGCCGTGTTC 1439 CATWDNTPSNIVVF 2263TGCGCAACATGGGATAACACGCCTTCGAATATTGTGGTATTC 1440 CATWDNTLSVWVF 2264TGCGCAACATGGGATAATACACTGAGTGTGTGGGTCTTC 1441 CATWDNTLSVNWVF 2265TGCGCAACATGGGATAACACCCTGAGTGTCAATTGGGTGTTC 1442 CATWDNTLNVFYVF 2266TGCGCAACCTGGGATAACACACTGAATGTCTTTTATGTTTTC 1443 CATWDNRLSSVVF 2267TGTGCGACATGGGATAATCGGCTCAGTTCTGTGGTCTTC 1444 CATWDNRLSAGVL 2268TGCGCAACATGGGATAACCGCCTGAGTGCTGGGGTGCTC 1445 CATWDNRLSAGVF 2269TGCGCAACGTGGGATAACAGGCTGAGTGCTGGGGTGTTC 1446 CATWDNRDWVF 2270TGCGCAACATGGGATAACAGGGATTGGGTCTTC 1447 CATWDNNLGAGVF 2271TGCGCAACATGGGATAACAACCTGGGTGCTGGGGTGTTC 1448 CATWDNKLTSGVF 2272TGCGCAACATGGGATAACAAGCTGACTTCTGGGGTCTTC 1449 CATWDNILSAWVF 2273TGCGCAACATGGGATAACATCCTGAGTGCCTGGGTGTTT 1450 CATWDNDIHSGLF 2274TGCGCAACCTGGGACAACGATATACATTCTGGGCTGTTC 1451 CATWDLSLSALF 2275TGCGCAACTTGGGATCTCAGCCTGAGTGCCCTGTTC 1452 CATWDITLSAEVF 2276TGCGCAACATGGGATATCACCCTGAGTGCTGAGGTGTTC 1453 CATWDISPSAGGVF 2277TGCGCAACGTGGGATATCAGCCCGAGTGCTGGCGGGGTGTTC 1454 CATWDISLSTGRAVF 2278TGCGCAACATGGGATATCAGTCTAAGTACTGGCCGGGCTGTGTTC 1455 CATWDISLSQVF 2279TGCGCAACATGGGATATCAGTCTGAGTCAGGTATTC 1456 CATWDIRLSSGVF 2280TGCGCAACATGGGATATCAGGCTGAGTAGTGGAGTGTTC 1457 CATWDIGPSAGGVF 2281TGCGCAACGTGGGATATCGGCCCGAGTGCTGGCGGGGTGTTC 1458 CATWDHSRAGVLF 2282TGCGCAACATGGGATCACAGCCGGGCTGGTGTGCTATTC 1459 CATWDHSPSVGEVF 2283TGCGCAACATGGGATCACAGTCCGAGTGTTGGAGAAGTCTTC 1460 CATWDHSLRVGVF 2284TGCGCAACATGGGATCACAGCCTGCGTGTTGGGGTGTTC 1461 CATWDHSLNIGVF 2285TGCGCAACATGGGATCACAGCCTGAACATTGGGGTGTTC 1462 CATWDHSLGLWAF 2286TGCGCAACATGGGATCACAGCCTGGGTCTTTGGGCATTC 1463 CATWDHNLRLVF 2287TGCGCCACATGGGATCACAATCTGCGTCTTGTTTTC 1464 CATWDHILASGVF 2288TGCGCGACTTGGGATCACATCCTGGCTTCTGGGGTGTTC 1465 CATWDFSLSVWVF 2289TGCGCAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1466 CATWDFSLSAWVF 2290TGCGCAACATGGGATTTCAGCCTGAGTGCTTGGGTGTTC 1467 CATWDDTLTAGVF 2291TGCGCAACATGGGATGACACCCTCACTGCTGGTGTGTTC 1468 CATWDDRLSAVLF 2292TGCGCAACATGGGACGACAGGCTGAGTGCTGTGCTTTTC 1469 CATWDDRLDAAVF 2293TGCGCAACATGGGATGACAGGCTGGATGCTGCGGTGTTC 1470 CATWDATLNTGVF 2294TGCGCAACATGGGATGCGACCCTGAATACTGGGGTGTTC 1471 CATWDASLSVWLL 2295TGCGCAACATGGGATGCCAGCCTGAGTGTTTGGCTGCTC 1472 CATWDASLSGGVF 2296TGCGCGACATGGGATGCCAGCCTGAGTGGTGGGGTGTTC 1473 CATRDTTLSAVLF 2297TGCGCAACACGGGATACCACCCTCAGCGCCGTTCTGTTC 1474 CATLGSSLSLWVF 2298TGCGCTACATTGGGTAGTAGCCTGAGTCTCTGGGTGTTC 1475 CATIETSLPAWVF 2299TGCGCAACAATCGAAACTAGCCTGCCTGCCTGGGTATTC 1476 CATGDRSLTVEVF 2300TGCGCAACAGGGGACAGAAGCCTGACTGTTGAGGTATTC 1477 CATGDLGLTIVF 2301TGCGCTACAGGGGATCTCGGCCTGACCATAGTCTTC 1478 CASWDYRGRSGWVF 2302TGCGCATCATGGGATTACAGGGGGAGATCTGGTTGGGTGTTC 1479 CASWDTTLNVGVF 2303TGCGCATCATGGGATACCACCCTGAATGTTGGGGTGTTC 1480 CASWDTTLGFVLF 2304TGCGCTTCATGGGATACCACCCTGGGTTTTGTGTTATTC 1481 CASWDTSLSGGYVF 2305TGCGCATCATGGGATACCAGCCTGAGTGGTGGTTATGTCTTC 1482 CASWDTSLRAGVF 2306TGCGCATCATGGGATACCAGCCTCCGTGCTGGGGTGTTC 1483 CASWDTSLGAGVF 2307TGCGCATCATGGGATACCAGCCTGGGTGCTGGGGTGTTC 1484 CASWDRGLSAVVF 2308TGCGCATCATGGGACAGAGGCCTGAGTGCAGTGGTGTTC 1485 CASWDNVLRGVVF 2309TGTGCTAGTTGGGATAACGTCCTGCGTGGTGTGGTATTC 1486 CASWDNRLTAVVF 2310TGCGCGTCATGGGATAACAGGCTGACTGCCGTGGTTTTC 1487 CASWDASLSVAF 2311TGCGCATCATGGGATGCAAGCCTGTCCGTCGCTTTC 1488 CASWDAGLSSYVF 2312TGCGCTTCGTGGGATGCCGGCCTGAGTTCTTATGTCTTC 1489 CASGDTSLSGVIF 2313TGCGCATCCGGGGATACCAGCCTGAGTGGTGTGATATTC 1490 CARWHTSLSIWVF 2314TGCGCAAGATGGCATACGAGCCTAAGTATTTGGGTCTTC 1491 CAIWDTGLSPGQVAF 2315TGCGCAATATGGGATACCGGCCTGAGTCCTGGCCAAGTTGCCTTC 1492 CAAWHSGLGLPVF 2316TGCGCAGCATGGCATAGCGGCCTGGGTCTCCCGGTCTTC 1493 CAAWDYSLSAGVF 2317TGCGCAGCATGGGATTACAGCCTGAGTGCTGGGGTGTTC 1494 CAAWDTTLRVRLF 2318TGCGCAGCCTGGGATACTACCCTGCGTGTTAGGCTGTTC 1495 CAAWDTSLTAWVF 2319TGCGCAGCATGGGATACCAGCCTGACTGCCTGGGTTTTC 1496 CAAWDTSLSGGVF 2320TGCGCAGCATGGGATACCAGCTTGAGTGGTGGGGTGTTC 1497 CAAWDTSLSGEAVF 2321TGCGCAGCATGGGATACCAGCCTGAGTGGCGAGGCTGTGTTC 1498 CAAWDTSLSGAVF 2322TGCGCAGCATGGGATACCAGCTTGAGTGGTGCGGTGTTC 1499 CAAWDTSLSAWVF 2323TGCGCAGCATGGGATACCAGCCTGAGTGCCTGGGTGTTC 1500 CAAWDTSLSAGVF 2324TGCGCAGCATGGGATACCAGCCTGAGTGCTGGGGTATTC 1501 CAAWDTSLDTYVF 2325TGCGCAGCATGGGATACCAGCCTGGATACTTATGTCTTC 1502 CAAWDTRLSGVLF 2326TGCGCTGCATGGGATACCCGTCTGAGTGGTGTGTTATTC 1503 CAAWDTRLSAGVF 2327TGCGCAGCATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1504 CAAWDRSLSTGVF 2328TGCGCAGCATGGGATCGCAGTCTGAGTACTGGAGTTTTC 1505 CAAWDIRRSVLF 2329TGCGCAGCGTGGGATATCCGCCGGTCTGTCCTTTTC 1506 CAAWDHTQRLSF 2330TGCGCTGCGTGGGATCACACTCAGCGTCTTTCCTTC 1507 CAAWDHSLSAGQVF 2331TGCGCAGCATGGGATCACAGCCTGAGTGCTGGCCAGGTGTTC 1508 CAAVDTGLKEWVF 2332TGCGCAGCAGTCGATACTGGTCTGAAAGAATGGGTGTTC

The CDRs were prescreened to contain no amino acid liabilities, crypticsplice sites or nucleotide restriction sites. The CDR variation wasobserved in at least two individuals and comprises the near-germlinespace of single, double and triple mutations. The order of assembly isseen in FIG. 12C.

The VH domains that were designed include IGHV1-69 and IGHV3-30. Each oftwo heavy chain VH domains are assembled with their respective invariant4 framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (H1, H2,H3) elements. For IGHV1-69, 417 variants were designed for H1 and 258variants were designed for H2. For IGHV3-30, 535 variants were designedfor H1 and 165 variants were designed for H2. For the CDR H3, the samecassette was used in both IGHV1-69 and IGHV-30 since both designed usean identical FW4, and because the edge of FW3 is also identical for bothIGHV1-69 and IGHV3-30. The CDR H3 comprises an N-terminus and C-terminuselement that are combinatorially joined to a central middle element togenerate 1×10¹⁰ diversity. The N-terminal and middle element overlapwith a “GGG” glycine codon. The middle and C-terminal element overlapwith a “GGT” glycine codon. The CDR H3 comprises 5 subpools that wereassembled separately. The various N-terminus and C-terminus elementscomprise sequences as seen in Table 10.

TABLE 10 Sequences for N-terminus and C-terminus elements SEQ ID ElementNO  Sequence Stem A 2333 CARDLRELECEEWT XXX SRGPCVDPRGVAGSFDVW Stem B2334 CARDMYYDF XXX EVVPADDAFDIW Stem C 2335 CARDGRGSLPRPKGGP XXXYDSSEDSGGAFDIW Stem D 2336 CARANQHF XXX GYHYYGMDVW Stem E 2337CAKHMSMQ XXX RADLVGDAFDVW

Example 8. GPCR Binding Protein Functionality

For a GPCR binding protein, the top 100-200 scFvs from phage-selectionswere converted to full-length immunoglobulins. After immunoglobulinconversion, the clones were transiently transfected in ExpiCHO toproduce immunoglobulins. Kingfisher and Hamilton were used for batch IgGpurifications followed by lab-chip to collect purity data for allpurified immunoglobulins. High yields and purities were obtained from 10mL cultures as seen in FIG. 13 and Table 11.

TABLE 11 Immunoglobulin Purity Percentage IgG % Name Purity mAb1  100mAb2  100 mAb3  100 mAb4  100 mAb5   98 mAb6  100 mAb7   97 mAb8  100mAb9  100 mAb10 100 mAb11 100 mAb12 100 mAb13 100 mAb14 100 mAb15 100

Stable cell lines expressing GPCR targets were then generated. FIG. 14shows target expression was confirmed by FACS. Cells expressing >80% ofthe target were then directly used for cell-based selections. Fiverounds of selections were carried out against cells overexpressingtarget of interest. 10⁸ cells were used for each round of selection.Before selection on target expressing cells, phage from each round wasfirst depleted on 10⁸ CHO background cells. Stringency of selections wasincreased by increasing the number of washes in subsequent rounds ofselection. Enrichment ratios were monitored for each round of selection.

Purified IgGs were tested for cell-binding affinity using FACS (FIGS.15A-15C) and cAMP activity (FIG. 15D). Allosteric inhibition wasobserved.

Purified IgGs were tested using BVP ELISA. As seen in FIG. 16 BVP ELISAshowed some clones comprising BVP scores comparable to comparatorantibodies.

Example 9: VHH Libraries

Synthetic VHH libraries were developed. For the ‘VHH Ratio’ library withtailored CDR diversity, 2391 VHH sequences (iCAN database) were alignedusing Clustal Omega to determine the consensus at each position and theframework was derived from the consensus at each position. The CDRs ofall of the 2391 sequences were analyzed for position-specific variation,and this diversity was introduced in the library design. For the ‘VHHShuffle’ library with shuffled CDR diversity, the iCAN database wasscanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's,1600 unique CDR2's, and 1608 unique CDR3's were identified and theframework was derived from the consensus at each framework positionamongst the 2391 sequences in the iCAN database. Each of the uniqueCDR's was individually synthesized and shuffled in the consensusframework to generate a library with theoretical diversity of3.2×10{circumflex over ( )}9. The library was then cloned in thephagemid vector using restriction enzyme digest. For the ‘VHH hShuffle’library (a synthetic “human” VHH library with shuffled CDR diversity),the iCAN database was scanned for unique CDRs in the nanobody sequences.1239 unique CDR1's, 1600 unique CDR2's, and 1608 unique CDR3's wereidentified and framework 1, 3, and 4 was derived from the human germlineDP-47 framework. Framework 2 was derived from the consensus at eachframework position amongst the 2391 sequences in the iCAN database. Eachof the unique CDR's was individually synthesized and shuffled in thepartially humanized framework using the NUGE tool to generate a librarywith theoretical diversity of 3.2×10{circumflex over ( )}9. The librarywas then cloned in the phagemid vector using the NUGE tool.

The Carterra SPR system was used to assess binding affinity and affinitydistribution for VHH-Fc variants. VHH-Fc demonstrate a range ofaffinities for TIGIT, with a low end of 12 nM K_(D) and a high end of1685 nM K_(D) (highlighted in top row; sixth from left-hand side andthird from right-hand side, respectively). Table 12 provides specificvalues for the VHH-Fc clones for ELISA, Protein A (mg/ml), and K_(D)(nM).

TABLE 12 ProA K_(D) Clone ELISA Library (mg/ml) (nM) 31-1  5.7 VHHhShuffle 0.29 12 31-6  9.6 VHH hShuffle 0.29 14 31-26 5.1 VHH hShuffle0.31 19 30-30 8.0 VHH Shuffle  0.11 23 31-32 8.0 VHH hShuffle 0.25 2729-10 5.0 VHH Ratio  0.19 32 29-7  7.3 VHH Ratio  0.28 41 30-43 13.5 VHH Shuffle  0.18 44 31-8  12.7  VHH hShuffle 0.29 45 31-56 11.7  VHHhShuffle 0.26 46 30-52 4.2 VHH Shuffle  0.22 49 31-47 8.8 VHH hShuffle0.23 53 30-15 9.3 VHH Shuffle  0.26 55 30-54 5.5 VHH Shuffle  0.30 5830-49 10.3  VHH Shuffle  0.26 62 29-22 3.4 VHH Ratio  0.27 65 29-30 9.2VHH Ratio  0.28 65 31-35 5.7 VHH hShuffle 0.24 66 29-1  10.4  VHH Ratio 0.09 68 29-6  6.8 VHH Ratio  0.29 69 31-34 6.0 VHH hShuffle 0.32 7029-12 6.2 VHH Ratio  0.23 70 30-1  5.4 VHH Shuffle  0.39 71 29-33 3.9VHH Ratio  0.15 74 30-20 4.6 VHH Shuffle  0.19 74 31-20 6.6 VHH hShuffle0.37 74 31-24 3.1 VHH hShuffle 0.15 75 30-14 9.9 VHH Shuffle  0.19 7530-53 7.6 VHH Shuffle  0.24 78 31-39 9.9 VHH hShuffle 0.32 78 29-1810.9  VHH Ratio  0.19 78 30-9  8.0 VHH Shuffle  0.40 79 29-34 8.6 VHHRatio  0.21 80 −29-27  8.6 VHH Ratio  0.18 82 29-20 5.9 VHH Ratio  0.2683 30-55 6.0 VHH Shuffle  0.41 85 30-39 6.1 VHH Shuffle  0.07 88 31-156.2 VHH hShuffle 0.32 88 29-21 4.3 VHH Ratio  0.23 88 29-37 5.3 VHHRatio  0.26 89 29-40 6.6 VHH Ratio  0.31 90 31-30 3.2 VHH hShuffle 0.3393 31-10 12.3  VHH hShuffle 0.31 94 29-3  13.6  VHH Ratio  0.11 94 30-575.2 VHH Shuffle  0.24 95 29-31 4.4 VHH Ratio  0.18 96 31-27 8.1 VHHhShuffle 0.31 96 31-33 6.0 VHH hShuffle 0.32 96 30-40 7.1 VHH Shuffle 0.21 99 31-18 4.1 VHH hShuffle 0.36 99 30-5  9.3 VHH Shuffle  0.05 100

Example 10. VHH Libraries for CRTH2R

A VHH library for CRTH2R was developed similar to methods described inExample 9. Briefly, stable cell lines expressing CRTH2R were generated,and target expression was confirmed by FACS. Cells expressing >80% ofthe target were then used for cell-based selections. Five rounds ofcell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage was amplified for the next roundof panning. A total of 1000 clones from round 4 and round 5 aresequenced by NGS to identify unique clones for reformatting as VHH-Fc.

26 binders out of the 175 unique CRTH2R VHH Fc binders had a target cellmean fluorescence intensity (MFI) value that was 2-fold over parentalcells. The data for variant CRTH2-41-51 is seen in FIGS. 17A-17B andTables 13A-13B. Tables 13A-13B show flow cytometry data as detected withthe RL1-A channel. Data for variant CRTH2-44-59 is seen in FIG. 18 andFIG. 19.

TABLE 13A Panning Summary VHH-Fc FACS binders Unique (MFI values 2 foldLibrary Phage over parental cells) VHH hShuffle 99 16 VHH Ratio/Shuffle76 10

TABLE 13B CRTH2-41-51 Data Sample Name Subset Name Count Median: RL1-ASample C7.fcs CRTH2R cells  8663 7441 Sample E10.fcs Parent Cells 115892120

Example 11. Identification of IgGs for CRTH2R

Cell binding of anti-CRTH2R antibodies was determined by testing on CHOCRTH2R-positive cells (GFP+) and parental CHO cells (GFP−), comparingparental negative and target positive cells to rule out false-positives.Antibodies as listed in Table 14A were titrated starting at 100 nM (15ug/mL) with 3-fold titrations, for a total of 8 points. Heavy and lightchain sequences for CRTH2R IgG antibodies are shown in Table 14B.Binding as detected by mean fluorescence intensity (MFI) byconcentration is shown in FIGS. 20A-20E. An exemplary gated dot plot andAPC histogram at 100 nM with CRTH2-27 is shown in FIGS. 21A-21B. Twoantibodies (gPCR-51 and gPCR-52) were used as a positive control.Binding profiles of the two positive controls are shown in FIGS.22A-22B.

TABLE 14A CRTH2R Antibody variable heavy and light chain sequences SEQID CRTH2R NO Antibody Heavy Chain 2338 CRTH2-74QVQLVESGGGWQPGRSLRLSCAASGFSFSEYGIHWVRQAPGKGLEWVAVISYEGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGYHYYGMDVWGQGTLV TVSS 2339CRTH2-24 QVQLVQSGAEVKKPGSSVKVSCKASGGSFSNYGISWVRQAPGQGLEWMGGIIPLIGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFTLGPQSIGPLGEVVPADDAFDIWGQGTLVTVSS 2340 CRTH2-28QVQLVQSGAEVKKPGSSVNVSCKASGGTFSDYAFSWVRQAPGQGLEWMGAIIPFFGTVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFATGTGGPEDDLYPQGELNDGYRIEVVPADDAFDIWGQGTLVTVSS 2341 CRTH2-39QVQLVQSGAEVKKPGSSVKVSCKASVDTFSRYSISWVRQAPGQGLEWMGGHPVFDTTNYAQKFQGRVTITADE5TSTAYMELSSLRSEDTAVYYCARDMYYDFGVILGGTAVGTNNGSANEVVPADDAFDIWGQGTLVTVSS 2342 CRTH2-19QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHAINWVRQAPGQGLEWMGRIIPIVGTTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFDYFGLTLTGDRNDDEVVPADDAFDIWGQGTLVTVSS 2343 CRTH2-9QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFWLGDQSTGSLIGAEVVPADDAFDIWGQGTLVTVSS 2344 CRTH2-8QVQLVQSGAEVKKPGSSVKVSCKASGGTFTDYAISWVRQAPGQGLEWMGGIIPFFGSPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFAAGLEGTITEVFDEEGHQGGTEVVPADDAFDIWGQGTLVTVSS 2345 CRTH2-27QVQLVESGGGVVQPGRSLRLSCAASGFTFDNYGMHWVRQAPGKGLEWVAVISYEGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFGSIYGEDWGELPEWPADDAFDIWGQGTLVTVSS 2346 CRTH2-45QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYAMHWVRQAPGKGLEWVADISHEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGRGSLPRPKGGPTSGGGFSTNIGYGFVVQSYDSSEDSGGAFDIWGQGTLVTVSS 2347 CRTH2-35QVQLVQSGAEVKKPGSSVKVSCKASGGTFRSYAISWVRQAPGQGLEWMGGIIPISGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFTRIFGNYQIYFGHFGYHYYGMDVWGQGTLVTVSS 2348 CRTH2-50QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYALSWVRKAPGQGLEWMGGTIPIFGTVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFTRVIGQPSPAVPSRGYIYHGYHYYGMDVWGQGTLVTVSS 2349 CRTH2-66QVQLVESGGGWQPGRSLRLSCAASGFDFSGYGMHWVRQAPGKGLEWVAVISYEGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRELECEEWTIEVHGQEFAVHQDRGGVFSRGPCVDPRGVAGSFDVWGQGTLVTVSS 2350 CRTH2-57QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGHPLFGTTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFVKIQGAPVSTPVPGFGTTGYHYYGMDVWGQGTLVTVSS 2351 CRTH2-32QVQLVESGGGWQPGRSLRLSCAASGFTFSKHGMHWVRQAPGKGLEWVAFISYEGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFHYSTVGATYYYYLGSETEVVPADDAFDIWGQGTLVTVSS 2352 CRTH2-15QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAIDWVRQAPGQGLEWMGGIIPLFGSPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFLYEGTSSSWLHVGHARYGYHYYGMDVWGQGTLVTVSS 2353 CRTH2-25QVQLVQSGAEVKKPGSSVKVSCKASGGSFRSYGISWVRQAPGQGLEWMGRIIPLFGTPDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFEDVDEGSLYLDMGRTFEVVPADDAFDIWGQGTLVTVSS 2354 CRTH2-42QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYAMHWVRQAPGKGLEWVAVISYEGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRELECEEWTVLQYGKFHMRWAESGEGSLSRGPCVDPRGVAGSFDVWGQGTLVTVSS 2355 CRTH2-55QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYDMHWVRQAPGKGLEWVAVISYEGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQASTEGDFGLEEVTGEGVDDRADLVGDAFDVWGQGTLVTVSS 2356 CRTH2-60QVQLVQSGAEVKKPGSSVKVSCKASGGTFKNYAINWVRQAPGQGLEWMGAIIPKFGAANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFSAVRGLAFGYGYRIGGYHYYGMDVWGQGTLVTVSS 2357 CRTH2-70QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNHAIIWVRQAPGQGLEWMGGIIPIFGTPSYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFDVISAGVVGAGNPEVVPADDAFDIWGQGTLVTVSS 2358 CRTH2-48-EVQLLESGGGLVQPGGSLRLSCAASGFSFSTHAMSWVRQAPGKGLEWVSTIGG 9SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAHGDSSSWYFSYYYMDVWGQGTLVTVSS 2359 CRTH2-41-EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAGISGS 51GGDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWG QGTLVTVSS 2360CRTH2-44- EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISMS 6GDDTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLRGRGDVSE YEYDWGQGTLVTVSSSEQ ID CRTH2R NO Antibody Light Chain 2361 CRTH2-74QSVLTQPPSVSAAPGQKVTJSCSGSTSNIGKNYVSWYQQLPGTAPKLLIYDDDERPSGIPDRFSGSMSGTSATLGITGLQTGDEADYYCEAWDADLSGAVFGGGTKLTVL 2362 CRTH2-24QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNFVSWYQQLPGTAPKLLIYDNIQRPSGIPDRFSGSKSGTSATLGITCLQTGDEADYYCGTWDTSLSAWFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2363 CRTH2-28QSVLTQPPSVSAAPGQKVTISCSGSISNIGKNYVSWYQQLPGTAPKLLIYDDHKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDRGLSAAVFGGGTKLTVL 2364 CRTH2-39QSVLTQPPSVSAAPGQKVTISCSGSSSNIGDNDVSWYQQLPGTAPKLLIYDDDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCASWDTSLSGGYVFGGGTKLTVL 2365 CRTH2-19QSALTQPASVSGSPGQSITISCTGTSSDVGGYDYVTWYQQHPGKAPKLMIYDVDTRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSTSYVFGGGTKLTVL 2366 CRTH2-9QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIYENDERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDTRLSAVVFGGGTKLTVL 2367 CRTH2-8QSVLTQPPSVSAAPGQKVTISCSGSSSNIGKNYVSWYQQLPGTAPKLLIYDNNQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLTSVVFGGGTKLTVL 2368 CRTH2-27QSALTQPASVSGSPGQSITISCTGTSNDVGAYNFVSWYQQHPGKAPKLMIYDISNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTRSNTRVFGGGTKLTVL 2369 CRTH2-45QSVLTQPPSVSAAPGQKVTISCSGTSSNIENNYVSWYQQLPGTAPKLLIYDNVKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNTVSAPWVFGGGTKLTVL 2370 CRTH2-35QSALTQPASVSGSPGQSITISCTGTSSDIGGYEFVSWYQQHPGKAPKLMJYGVSRRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTPYVFGGGTKLTVL 2371 CRTH2-50QSALTQPASVSGSPGQSITISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDVSNRPQGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSNTYWFGGGTKLTVL 2372 CRTH2-66EIVMTQSPATLSVSPGERATLSCRASQGVGSNLAWYQQKPGQAPRLLIYRTSIRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYYSWPPLTFGGGTKVEIK 2373 CRTH2-57QSVLTQPPSVSAAPGQKVTISCSGSSSNIEDNYVSWYQQLPGTAPKLLIYDNFKRPGSIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSAALFGGGTKLTVL 2374 CRTH2-32QSALTQPASVSGSPGQSITISCTGTSSGVGGYDYVSWYQQHPGKAPKLMIYDDNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTGSSTLYVFGGGTKLTVL 2375 CRTH2-15QSVLTQPPSVSAAPGQKVTISCSGSGSNIGSNYVSWYQQLPGTAPKLLIYDNIRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCAAWDTRLSAGVFGGGTKLTVL 2376 CRTH2-25DIQMTQSPSSLSASVGDRVTITCRASQGISTYLNWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP-WTFGGGTKVEIK 2377 CRTH2-42QSALTQPASVSGSPGQSITISCTGTSSDVGGYRYVSWYQQHPGKAPKLMIYNVNYRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYRSSSTLGVFGGGTKLTVL 2378 CRTH2-55QSVLTQPPSVSAAPGQKVTISCSGSSSNIGDNFVSWYQQLPGTAPKLLIYDDDERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDRSLSAVVFGGGTKLTVL 2379 CRTH2-60QSVLTQPPSVSAAPGQKVTISCSGSTSNIGINYVSWYQQLPGTAPKLLIYENRKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDASLKNLVFGGGTKLTVL 2380 CRTH2-70QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNFVSWYQQLPGTAPKLLIYDNEKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDERQTDESYVFGGGTKLTVL 2381 CRTH2-48-DIQMTQSPSSLSASVGDRVTITCRASQSISDYVNWYQQKPGKAPKLLIYGASILQT 9GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFTTPWTFGGGTKVEIK

TABLE 14B Variably Heavy Chain CDR3 Sequences SEQ CRTH2R ID NO AntibodyCDRH3 2382 CRTH2-74 CARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGY HHYYGMDVW 2383 CRTH2-24CARDMYYDFTLGPQSIGPLGEVVPADD AFDIW 2384 CRTH2-28CARDMYYDFATGTGGPEDDLYPQGELN DGYRIEVVPADDAFDIW 2385 CRTH2-39CARDMYYDFGVILGGTAVGTNNGSANE VVPADDAFDIW 2386 CRTH2-19CARDMYYDFDYFGLTLTGDRNDDEVVP ADDAFDIW 2387 CRTH2-9CARDMYYDFWLGDQSTGSLIGAEVVPA DDAFDIW 2388 CRTH2-8CARDMYYDFAAGLEGTITEVFDEEGHQ GGTEWPADDAFDIW 2389 CRTH2-27CARDMYYDFGSIYGEDVVGELPEVVPA DDAFDIW 2390 CRTH2-45CARDGRGSLPRPKGGPTSGGGFSTNIG YGFWQSYDSSEDSGGAFDIW 2391 CRTH2-35CARANQHFTRIFGNYQIYFGHFGYHYY GMDVW 2392 CRTH2-50CARANQHFTRVIGQPSPAVPSRGYIYH GYHYYGMDVW 2393 CRTH2-66CARDLRELECEEWTIEVHGQEFAVHQD RGGVFSRGPCVDPRGVAGGSFDVW 2394 CRTH2-57CARANQHFVKIQGAPVSTPVPGFGTTG YHYYGMDVW 2395 CRTH2-32CARDMYYDFHYSTVGATYYYYLGSETE VVPADDAFDIW 2396 CRTH2-15CARANQHFFLYEGTSSSWLHVGHARYG YHYYYGMDVW 2397 CRTH2-25CARDMYYDFEDVDEGSLYLDMGRTFEV VPADDAFDIW 2398 CRTH2-42CARDLRELECEEWTVLQYGKFHMRWAE SGEGSLSRGPCVDPRGVAGSFDVW 2399 CRTH2-55CAKHMSMQASTEGDFGLEEVTGEGVDD RADLVGDAFDVM 2400 CRTH2-60CARANQHFSAVRGLAFGYGYRIGGYHY YGMDVW 2401 CRTH2-70CARDMYYDFDVISAGVVGAGNPEVVPA DDAFDIW 2402 CRTH2-74CARDMYYDFDVISAGVVGAGNPEVVPA DDAFDIW

In subsequent examples, five antibodies were shown to have functionaleffects in cAMP assays; CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, andCRTH2-42. The binding curves of these antibodies are compared in FIGS.23A-23B.

Example 12. Antagonist Activity Using cAMP Assay

A library of CRTH2R IgG antibodies were assayed to determine antagonistfunction in PGD2-induced cAMP signals. Briefly, cells were pre-incubatedwith IgG (titration 1:3) for 1 hour at room temperature. Subsequently,cells were stimulated with PGD2 (0.59 nM) for 30 min at 37° C. in thepresence of forskolin, since CRTH2R is Gα₁ coupled.

Results showing effect of antibody on detected signal in relative lightunits (rlu) are shown in. At the highest concentration tested (300 nM),some of the CRTH2R IgGs caused an upward deflection of the signal,indicating inhibition of the cAMP signal induced by PGD2 stimulation.For comparison, bar charts showing the ratio of IgG treated versuscontrol treated for the three highest IgG concentrations tested areshown in FIG. 24A. Antibodies depicted in FIG. 24B show CRTH2R IgGantibodies which resulted in more than a 20% antagonist activity at 33nM, specifically CRTH2-74, CRTH2-24, CRTH2-28, CRTH2-19, CRTH2-45,CRTH2-9, CRTH2-8, CRTH2-15, CRTH2-42, CRTH2-60, and CRTH2-70.

Example 13. Allosteric Modulation of PGD2-Induced cAMP Signal

CRTH2R IgG antibodies were assayed for allosteric activity. Allostericmodulation was determined by assaying CRTH2R IgG antibodies inPGD2-induced cAMP signal. Briefly, cells were re-incubated with no IgGantibody or 100 nM CRTH2R IgG antibody. Subsequently, cells werestimulated with PGD2 at various concentrations in the presence offorskolin followed by assay for cAMP activity.

Results of the cAMP assays is seen in FIG. 25. A right-ward shift thePGD2 dose response curve (and increase in IC50 value) indicates anegative allosteric effect. As shown in FIG. 25, five of the CRTH2R IgG(CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, and CRTH2-42) caused an IC50fold difference of >2.0 compared with PGD2 alone, suggesting they arenegative allosteric modulators.

Example 14. Agonist Activity of PGD2-Induced cAMP Signal

CRTH2R IgG antibodies were assayed for agonist function. Agonistactivity was determined by assaying CRTH2R IgG antibodies described inExample 11 in PGD2-induced cAMP signal.

Briefly, cells were treated with PGD2 or CRTH2R IgG antibodies both inthe presence of forskolin. The CRTH2R IgG antibodies included CRTH2-74,CRTH2-24, CRTH2-28, CRTH2-39, CRTH2-19, CRTH2-9, CRTH2-8, CRTH2-27,CRTH2-45, CRTH2-35, CRTH2-50, CRTH2-66, CRTH2-57, CRTH2-32, CRTH2-15,CRTH2-25, CRTH2-42, CRTH2-55, CRTH2-60, and CRTH2-70. Treatmentstimulations were performed for 30 min at 37° C. cAMP assays were thenperformed (data not shown).

Example 15. Control Experiments Showing Allosteric Modulators

Allosteric modulation was determined for a known CRTH2R antagonist(small molecule 00000459) and two control antibodies. Experiments wereperformed similar to those described in Example 13. Briefly, cells weretreated with OC000459, comparator CRTH2R AB51 antibody, or comparatorCRTH2R AB52 antibody. Cells were then stimulated with PGD2 in thepresence of forskolin.

Results are shown in FIGS. 26A-26C. OC000459 causes a strong right-wardshift of the curve and a 459-fold increase in the IC50 value (FIG. 26A).Incubation with CRTH2R AB51 caused no change in IC50 value (FIG. 26B).Incubation with the comparator antibody #52 caused a 3.5-fold decreasein the IC50 value, indicating it is a positive allosteric modulator,i.e. it has agonistic effects (FIG. 26C).

Example 16. CRTH2R β-Arrestin Recruitment Assay for AntagonistModulation

Antagonist modulation by nine CRTH2R IgG antibodies was determined. Thenine CRTH2R IgG antibodies included CRTH2-9, CRTH2-27, CRTH2-50,CRTH2-32, CRTH2-42, CRTH2-74, CRTH2-55, CRTH2-28, and CRTH2-39. Theantagonist function of these nine antibodies as compared to 00000459 wasdetermined using a PGD2-induced β-arrestin recruitment. Results,including a positive control using small molecule OC000459, are shown inFIGS. 27A-27D.

Example 17. CRTH2R β-Arrestin Recruitment Assay for AllostericModulation

Allosteric modulation by nine CRTH2R IgGs were determined. The nineCRTH2R IgGs included CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, CRTH2-42,CRTH2-74, CRTH2-55, CRTH2-28, and CRTH2-39. The allosteric modulation ofthese nine antibodies as compared to OC000459 was determined using aPGD2-induced β-arrestin recruitment.

Briefly, cells were pre-incubated with IgG (100 nM) for 1 hour at roomtemperature followed by PGD2 stimulation for 90 min at 37° C. Data wasnormalized against the first data point (lowest PGD2 and zero Ab) ineach graph. Results, including a positive control using small moleculeOC000459, are shown in FIGS. 27A-27D.

Example 18. CRTH2R Hyperimmune Immunoglobulin Library

A hyperimmune immunoglobulin (IgG) library was created using similarmethods as described in Example 9. Briefly, the hyperimmune IgG librarywas generated from analysis of databases of human naïve and memoryB-cell receptor sequences consisting of more than 37 million unique IgHsequences from each of 3 healthy donors. More than two million CDRH3sequences were gathered from the analysis and individually constructedusing methods similar to Examples 1-3. Any duplicate CDRH3s andpotential liability motifs that frequently pose problems in developmentwere removed during the library synthesis step. These CDRH3 sequencediversities were then combinatorially assembled and incorporated ontothe DP47 human framework to construct a highly functional antibody Fablibrary with 1×10¹⁰ size. A schematic of the design can be seen in FIG.28.

A CRTH2R hyperimmune immunoglobulin library was generated. Briefly, fiverounds of cell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage gets amplified for the nextround of panning.

CRTH2R immunoglobulins were assessed for binding affinity and allostericmodulator function of PGD2-induced cAMP. As seen in FIGS. 29A-29F, threespecific CRTH2R immunoglobulins were identified with sub nanomolar tosingle digit nanomolar cell binding affinities to hCRTH2R and hadinhibitory activities in the allosteric cAMP assay. The sequences forthe three CRTH2R immunoglobulins CRTH2-48-3, CRTH2-48-21, andCRTH2-48-27 are seen in Table 15.

TABLE 15 CRTH2R sequences SEQ ID NO: IgG Amino Acid SequenceVariable Heavy Chain 2403 CRTH2-48- EVQLVESGGGLVQAGGSLRLSCAASGS 3IFRINAMGWFRQAPGKEREGVAAINNF GTTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDRY DWGQGTQVTVSS 2404 CRTH2-48-EVQLVESGGGLVQAGGSLRLSCAASGS 21 FFSINAMGWFRQAPGKEREFVAGITRSGVSTSYADSVKGRFTISADNAKNTVYL QMNSLKPEDTAVYYCAAHRIVVGGTSV GDWRWGQGTQVTVSS2405 CRTH2-48- EVQLVESGGGLVQAGGSLRLSCAASGS 27IFHINAMGWFRQAPGKEREGVAAINNF GTTKYADSVKGRFTISANNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDR YDWGQGTLVTVSS Variable Light Chain 2406CRTH2-48- DIQMTQSPSSLSASVGDRVTITCRASQ 3 SISSDLNWYQQKPGKAPKLLIYFASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQSYSSPLTFGGGTKVEIKR 2407 CRTH2-48-DIQMTQSPSSLSASVGDRVTITCRTSQ 21 SISNYLNWYQQKPGKAPKLLIYATSSLESGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQSYSTLLTFGGGTKVEIKR 2408 CRTH2-48-DIQMTQSPSSLSASVGDRVTITCRASQ 27 SISRYLHWYQQKPGKAPKLLIYGASRLESGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCRQSYSTPWTFGGGTKVEIKR 

Example 19. GPCR Libraries with Varied CDR's

A GPCR library was created using a CDR randomization scheme.

Briefly, GPCR libraries were designed based on GPCR antibody sequences.Over sixty different GPCR antibodies were analyzed and sequences fromthese GPCRs were modified using a CDR randomization scheme.

The heavy chain IGHV3-23 design is seen in FIG. 30A. As seen in FIG.30A, IGHV3-23 CDRH3's had four distinctive lengths: 23 amino acids, 21amino acids, 17 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 23 amino acids in length, 30% for the CDRH321 amino acids in length, 20% for the CDRH3 17 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9.3×10⁸, and the full heavy chain IGHV3-23 diversitywas 1.9×10¹³.

The heavy chain IGHV1-69 design is seen in FIG. 30B. As seen in FIG.30B, IGHV1-69 CDRH3's had four distinctive lengths: 20 amino acids, 16amino acids, 15 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 20 amino acids in length, 30% for the CDRH316 amino acids in length, 20% for the CDRH3 15 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9×10⁷, and the full heavy chain IGHV-69 diversity is4.1×10¹².

The light chains IGKV 2-28 and IGLV 1-51 design is seen in FIG. 30C.Antibody light chain CDR sequences were analyzed for position-specificvariation. Two light chain frameworks were selected with fixed CDRlengths. The theoretical diversities were determined to be 13800 and5180 for kappa and light chains, respectively.

The final theoretical diversity was determined to be 4.7×10¹⁷ and thefinal, generated Fab library had a diversity of 6×10⁹. See FIG. 30D.

Example 20. CRTH2R Libraries with Varied CDR's

A CRTH2R library is created using a CDR randomization scheme similarlydescribed in Example 19.

Briefly, CRTH2R libraries are designed based on GPCR antibody sequences.Over sixty different GPCR antibodies are analyzed and sequences fromthese GPCRs are modified using a CDR randomization scheme. CRTH2Rvariant IgGs designed using the CDR randomization scheme are purifiedand are assayed to determine cell-based affinity measurements and forfunctional analysis.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. A nucleic acid library, comprising: a plurality of nucleic acids,wherein each of the nucleic acids encodes for a sequence that whentranslated encodes for a CRTH2R binding immunoglobulin, wherein theCRTH2R binding immunoglobulin comprises a variant of a CRTH2R bindingdomain, wherein the CRTH2R binding domain is a ligand for the CRTH2R,and wherein the nucleic acid library comprises at least 10,000 variantimmunoglobulin heavy chains and at least 10,000 variant immunoglobulinlight chains.
 2. The nucleic acid library of claim 1, wherein thenucleic acid library comprises at least 50,000 variant immunoglobulinheavy chains and at least 50,000 variant immunoglobulin light chains. 3.(canceled)
 4. The nucleic acid library of claim 1, wherein the nucleicacid library comprises at least 10⁵ non-identical nucleic acids.
 5. Thenucleic acid library of claim 1, wherein a length of the immunoglobulinheavy chain when translated is about 90 to about 100 amino acids.
 6. Thenucleic acid library of claim 1, wherein a length of the immunoglobulinheavy chain when translated is about 100 to about 400 amino acids. 7.The nucleic acid library of claim 1, wherein the variant immunoglobulinheavy chain when translated comprises at least 80% sequence identity toany one of SEQ ID NO: 2338-2360 or 2403-2405.
 8. The nucleic acidlibrary of claim 1, wherein the variant immunoglobulin light chain whentranslated comprises at least 80% sequence identity to any one of SEQ IDNO: 2361-2381 or 2406-2408.
 9. A nucleic acid library comprising aplurality of nucleic acids, wherein each nucleic acid of the pluralityof nucleic acids encodes for a sequence that when translated encodes foran antibody or antibody fragment thereof, wherein the antibody orantibody fragment thereof comprises a variable region of a heavy chain(VH) that comprises a CRTH2R binding domain, wherein each nucleic acidof the plurality of nucleic acids comprises a sequence encoding for asequence variant of the CRTH2R binding domain, and wherein the antibodyor antibody fragment binds to its antigen with a K_(D) of less than 100nM.
 10. The nucleic acid library of claim 9, wherein a length of the VHis about 90 to about 100 amino acids.
 11. The nucleic acid library ofclaim 9, wherein a length of the VH is about 100 to about 400 aminoacids.
 12. (canceled)
 13. (canceled)
 14. The nucleic acid library ofclaim 9, wherein the library comprises at least 10⁵ non-identicalnucleic acids. 15.-20. (canceled)
 21. An antibody or antibody fragmentthat binds CRTH2R, comprising an immunoglobulin heavy chain and animmunoglobulin light chain: a. wherein the immunoglobulin heavy chaincomprises an amino acid sequence at least about 90% identical to thatset forth in any one of SEQ ID NO: 2338-2360 or 2403-2405; and b.wherein the immunoglobulin light chain comprises an amino acid sequenceat least about 90% identical to that set forth in any one of SEQ ID NO:2361-2381 or 2406-2408. 22.-45. (canceled)
 46. The antibody or antibodyfragment of claim 21, wherein the antibody is a monoclonal antibody, apolyclonal antibody, a bi-specific antibody, a multispecific antibody, agrafted antibody, a human antibody, a humanized antibody, a syntheticantibody, a chimeric antibody, a camelized antibody, a single-chain Fvs(scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, aFd fragment, a Fv fragment, a single-domain antibody, an isolatedcomplementarity determining region (CDR), a diabody, a fragmentcomprised of only a single monomeric variable domain, disulfide-linkedFvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or abantigen-binding fragments thereof.
 47. The antibody or antibody fragmentof claim 21, wherein the antibody or antibody fragment thereof ischimeric or humanized.
 48. (canceled)
 49. (canceled)
 50. The antibody orantibody fragment of claim 21, wherein the antibody has an EC50 lessthan about 10 nanomolar in a cAMP assay.
 51. The antibody or antibodyfragment of claim 21, wherein the antibody or antibody fragmentcomprises a complementarity determining region (CDR) comprising an aminoacid sequence at least about 90% identical to that set forth in any oneof SEQ ID NOs: 2382-2402.
 52. (canceled)
 53. A method of treating adisease or disorder of the central nervous system, kidney, intestine,lung, hair, skin, bone, or cartilage, comprising administering theantibody or antibody fragment of claim
 21. 54. A method of treating adisease or disorder characterized by an inflammatory response,comprising administering the antibody or antibody fragment of claim 21.55. A method of treating an allergic reaction, comprising administeringthe antibody or antibody fragment of claim
 21. 56. The method of claim55, wherein the allergic reaction is chronic idiopathic urticaria orallergic rhinitis. 57.-77. (canceled)